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.

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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Research

JoVE Journal

Neuroscience

885 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

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.

Thorough preclinical testing of drugs that act in the central nervous system often involves assessing and comparing drug biodistribution in association with specific routes of administration. Here, three commonly used methods of systemic delivery (intravenous, intraperitoneal, and oral) as well as a method for local delivery (convection-enhanced delivery) are demonstrated in mice.

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. Understanding repair and regeneration in animals is a key question in biology. The damaged human CNS does not regenerate, and understanding how to promote the regeneration is one of main goals of medical neuroscience. The powerful genetic toolkit of Drosophila can be used to tackle the problem of CNS regeneration.
A lesion to the CNS ventral nerve cord (VNC, equivalent to the vertebrate spinal cord) is applied manually with a tungsten needle. The VNC can subsequently be filmed in time-lapse using laser scanning confocal microscopy for up to 24 hr to follow the development of the lesion over time. Alternatively, it can be cultured, then fixed and stained using immunofluorescence to visualize neuron and glial cells with confocal microscopy. Using appropriate markers, changes in cell morphology and cell state as a result of injury can be visualized. With ImageJ and purposely developed plug-ins, quantitative and statistical analyses can be carried out to measure changes in wound size over time and the effects of injury in cell proliferation and cell death. These methods allow the analysis of large sample sizes. They can be combined with the powerful genetics of Drosophila to investigate the molecular mechanisms underlying CNS regeneration and repair.

An Injury Paradigm to Investigate Central Nervous System Repair in Drosophila

An injury paradigm using the Drosophila larval ventral nerve cord to investigate central nervous system regeneration and repair is described. Stabbing followed by laser scanning confocal microscopy in time-lapse and fixed specimens, combined with quantitative analysis with purposefully developed software and genetics, are used to investigate the molecular mechanisms of CNS regeneration and repair.

An experimental method has been developed to investigate the cellular responses to central nervous system (CNS) injury using the fruit-fly Drosophila. ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...