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

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</ol>

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><tbody><tr><td>1-Pentanol</td><td>Fisher Scientific</td><td>AC160600250</td><td>Used to make Avertin solution&amp;nbsp;</td></tr><tr><td>2,2,2-tribomoethanol</td><td>Sigma Aldrich</td><td>T48402</td><td>Used to make Avertin solution&amp;nbsp;</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&amp;nbsp;</td><td>Fisher Scientific</td><td>A451-1&amp;nbsp;</td><td></td></tr><tr><td>50 kS/s/channel Bridge Analog Input Module&amp;nbsp;</td><td>National Instruments</td><td>NI-9237</td><td>DAQ module</td></tr><tr><td>Albumin Bovine Fraction V (BSA)&amp;nbsp;</td><td>Research Products International&amp;nbsp;</td><td>A30075</td><td>BSA</td></tr><tr><td>Anti-Iba1 Primary Antibody (Goat polyclonal)&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>ab5076</td><td>Marker for microglia, Used at 1:500 concentration&amp;nbsp;</td></tr><tr><td>Anti-Synaptophysin Primary Antibody (Mouse monoclonal)&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>ab8049</td><td>Marker for photoreceptor ribbon synapses, Used at 1:20 concentration</td></tr><tr><td>Araldite GY 502&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Used to refill pressurized air tank</td></tr><tr><td>DAPI Fluoromount-G&amp;nbsp;</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.&amp;nbsp;</td><td></td><td>For sectioning optic nerves, 3 mm/45 degrees/Style H&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Donkey Serum&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Ethanol (200 proof)&amp;nbsp;</td><td>KOPTEC (Supplier: VWR)&amp;nbsp;</td><td>89125-188</td><td>Ethanol&amp;nbsp;</td></tr><tr><td>Fluoromount-G&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma Aldrich</td><td>322415-2L&amp;nbsp;</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 Measurement and Automation Explorer (NI MAX)&amp;nbsp;</td><td>National Instruments</td><td></td><td>Software to configure DAQ system settings</td></tr><tr><td>NI-DAQmx drivers&amp;nbsp;</td><td>National Instruments</td><td></td><td>Driver for interacing with DAQ system&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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)&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>ab32376</td><td>Marker for rod bipolar cells, Used at 1:500 concentration&amp;nbsp;</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&amp;nbsp;</td><td>Honeywell&amp;nbsp;</td><td>060-0708-10TJG</td><td>Consider the range when selecting pressure transducer to optimize resolution of measurements&amp;nbsp;</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S5016</td><td></td></tr><tr><td>Super TJE Pressure Transducer, 7500 psi&amp;nbsp;</td><td>Honeywell&amp;nbsp;</td><td></td><td>Consider the range when selecting pressure transducer to optimize resolution of measurements&amp;nbsp;</td></tr><tr><td>Syringe/Needle Combo</td><td>Covetrus&amp;nbsp;</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&amp;nbsp;</td><td>Freezing medium&amp;nbsp;</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&amp;nbsp;</td><td>AXY40 Series</td><td>XY positioning table&amp;nbsp;</td></tr><tr><td>University Brush - Series 233- Round, Size 000</td><td>Winsor and Newton&amp;nbsp;</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&amp;nbsp;</td><td></td><td>Digital tool for data acquisition software&amp;nbsp;</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.

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>

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

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.

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 ...

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

Universidad Científica del Sur

Research

JoVE Journal

Neuroscience

1.0K 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. 

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

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

A Novel Vertebral Stabilization Method for Producing  Contusive Spinal Cord Injury

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing reliable cervical SCI is difficult due to lack of satisfactory methods of vertebral stabilization. The conventional method to stabilize the spine is to suspend the rostral and caudal cervical spine via clamps attached to cervical spinous processes. &#160;However, this method of stabilization fails to prevent tissue yielding during the contusion as the cervical spinal processes are too short to be effectively secured by the clamps (Figure 1). &#160;Here we introduce a new method to completely stabilize the cervical vertebra at the same level of the impact injury. &#160;This method effectively minimizes movement of the spinal column at the site of impact, which greatly improves the production of consistent SCIs. &#160;We provide visual description of the equipment (Figure 2-4), methods, and a step-by-step protocol for the stabilization of the cervical 5 vertebra (C5) of adult rats, to perform laminectomy (Figure 5) and produce a contusive SCI thereafter. &#160;Although we only demonstrate a cervical hemi-contusion using the NYU/MASCIS impactor device, this vertebral stabilization technique can be applied to other regions of the spinal cord, or be adapted to other SCI devices. &#160;Improving spinal cord exposure and fixation through vertebral stabilization may be valuable for producing consistent and reliable injuries to the spinal cord. &#160;This vertebral stabilization method can also be used for stereotactic injections of cells and tracers, and for imaging using two-photon microscopy in various neurobiological studies.

Vertebral stabilization is necessary for minimizing variability, and for producing consistent experimental spinal cord injuries.&#160; Using a customized stabilizing apparatus in conjunction with the NYU/MASCIS impactor device, we have demonstrated here the proper equipment and procedure for generating reproducible hemi-contusive cervical (C5) spinal cord injuries in adult rats.

Clinically-relevant animal cervical spinal cord injury (SCI) models are essential for developing and testing potential therapies; however, producing ...

Medicine

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.

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

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic brain injury. While elevations in ICP are common, data regarding the measurement and treatment of these ICP elevations are conflicting. There is increasing recognition that changes in the balance between supply and demand of brain tissue are critically important and therefore the measurement of multiple modalities is required. Approaches are not standard, and therefore this article provides a description of a bedside, single burr hole approach to multimodality monitoring that allows the passage of probes designed to measure not only ICP but brain tissue oxygen, blood flow, and intracranial electroencephalography. Patient selection criteria, operative procedures, and practical considerations for securing probes during critical care are described. This method is readily performed, safe, secure, and flexible for the adoption of a variety of multimodality monitoring approaches aimed at detecting or preventing secondary brain injuries.

A method of recording multimodality monitoring signals in patients with severe brain injuries using a bedside, single burr hole technique is described.

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

Induction of Traumatic Brain Injury in a Rat Model Using a Lateral Fluid Percussion Device

Make a 1.5 to 2.5-centimeters midline incision through the skin and muscle of the scalp using a number 10 scalpel blade. Retract the skin and muscle to expose the skull and provide a clear surgical field. Electric cautery is useful for achieving quick hemostasis.
Next, shave down the lateral ridge of the left parietal bone, using a surgical curette to produce a smooth, flat surface so that the base of the female-female Luer Lock hub can rest flush with the skull. Irrigate the skull surface and surrounding tissues with 2.0 mg per milliliter gentamicin solution in sterile saline, and blot excess solution with sterile swabs. Then apply 3% hydrogen peroxide to the skull to dry the bone.
At this point, create a 5-millimeter diameter craniectomy site through the left parietal bone. Then, remove the bone flap with the surgical curette and smooth tissue forceps.
Next, using a stereomicroscope, gently remove the thin rim of bone remaining with smooth tissue forceps taking care not to rupture the dura. Then swab the skull with 70% ethanol to remove any bone dust, and to dry the skull. Apply a thin layer of cyanoacrylate glue around the bottom edge of the Luer Lock hub, and secure it to the skull over the craniectomy without obstructing the opening and without allowing the glue to contact the dura. Then seal the Luer lock in place with a thin layer of glue around the outside base of the hub.
Next, prepare a slurry of dental cement and apply this to the surface of the skull around and over the base of the Luer Lock hub to secure it in place. Then fill the Luer Lock hub with a sterile, preservative-free solution containing multiple electrolytes using a syringe and needle. A convex bolus of saline should be seen above the top of the rim. Once the dental cement is completely cured, discontinue gas anesthesia and remove the rat from the stereotaxic frame.
Place the rat on a platform next to the fluid percussion injury device in sternal recumbency. Then, secure a 12-centimeter length of pressure tubing to the end of the device&#39;s curved tip, with the opposite end terminating in a 2-centimeter male Luer Lock twist connector. Secure the rat to the device by connecting the female end of the hub on the rat skull to the male connector.
Repeatedly check the animal for return of withdrawal reflex. As soon as the rat regains withdrawal reflex, but is still sedated, release the pendulum of the device to cause a single 20-millisecond pressure pulse and induce injury. Then immediately disconnect the rat from the FPI device, place it in sternal recumbency, and provide supplemental oxygen via a nose cone until spontaneous breathing returns.
Note that apnea is an anticipated consequence of the injury. If necessary, provide periodic manual breaths via a bag valve mask until the rat begins to spontaneously breathe on its own. Monitor continuously and record the time of return of righting reflex.