This work was supported by two grants from JSPS KAKENHI (Grant Numbers 21K09573 (K.M.) and 23K15901 (T.K.)).

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Defense Medical College (approval #18050) and performed in accordance with the guidelines of the National Institutes of Health and the Ministry of Education, Culture, Sports, Science, and Technology of Japan. All efforts were made to minimize the number of animals and their suffering.
1. Animals
<ol>
	<li>Use 8-week-old male CBA/J mice to follow this protocol. Before the experiment, subject the mice to a hearing function test and endoscopic observation of the tympanic membrane to ensure normality.</li>
	<li>Divide 27 CBA/J mice into three groups: (1) 2.0 J/cm2 exposed group (n = 9 mice); (2) 2.25 J/cm2 exposed group (n = 9 mice); and (3) 2.5 J/cm2 exposed group (n = 9 mice). Remove all the ears for assessment 1 month after the LISW exposure.</li>
</ol>
2. Experimental settings of LISW exposure
<ol>
	<li>The laser target is a black, natural rubber disk, 10 mm in diameter and 0.5 mm thick. To increase the LISW impulse, use an acrylic resin welding adhesive to bond a 1.0 mm thick, transparent, polyethylene terephthalate sheet (PET) to the top of the target area. Irradiate a 532 nm Q-switched Nd: YAG laser to generate the LISW behind the target (Figure 1A).</li>
	<li>Focus the laser pulse with a plano-convex lens to a 3.0 mm diameter spot on the laser target.</li>
	<li>Use the LISW irradiation to generate plasma at the bonding surface of the two materials and vaporize the rubber (plasma-mediated ablation), leaving vaporized rubber in the cavity.</li>
	<li>Use a hydrophone to measure the pressure wave of LISW at 1.0 mm underwater, not in living tissue. Place a 0.25 mm diameter fiber optic hydrophone under the black rubber 1.0 mm below the water surface to record the LISW pressure waveforms and measure them using a digital oscilloscope. 
	NOTE: The pressure waveforms showed stable characteristics with similar maximum pressure and impulse as shown in Figure 1B.</li>
	<li>Perform all animal procedures under general anesthesia using intraperitoneal injections of 1 mg/kg medetomidine hydrochloride and 75 mg/kg ketamine. Apply an ophthalmic ointment to both eyes of the mouse to prevent drying and provide heat support.</li>
	<li>Carefully shave the postauricular regions to avoid retaining the trapped air in the fur. Fix the mice on a plate and position the postauricular regions in the focal area of the LISW in a vertically upward direction.</li>
	<li>Attach a black rubber target percutaneously to the postauricular region of the mouse ear. To ensure acoustic impedance matching, use an ultrasound conductive gel between the laser target and the skin surface.</li>
	<li>Apply a single LISW pulse to the cochlea via the temporal bone. Set the outputs of the laser pulses to three energy densities: 2.0 J/cm2 , 2.25 J/cm2, and 2.5 J/cm2.</li>
</ol>
3. Cochlear function test
NOTE: Auditory brainstem response (ABR) tests were performed as previously reported14,15.
<ol>
	<li>Perform the ABR measurement 1 day before and 1 day and 1 month after LISW exposure.</li>
	<li>ABR is an auditory evoked potential in response to auditory stimuli and is commonly used to assess hearing thresholds at four frequencies (12.0 kHz, 16.0 kHz, 20.0 kHz, and 24.0 kHz).</li>
	<li>Present the stimulation sound over a small earphone and measure the sound pressure level near the tympanic membrane of the mouse using a small microphone placed near the earphone. Output burst stimuli from a sound generator at 37 cycles/s and amplify the sound pressure from a 20 dB sound pressure level (SPL) to 80 dB SPL in 5 dB SPL steps.</li>
	<li>Insert a stainless steel needle electrode for electroencephalogram recording under the ear canal and frontal region of the ear and insert a ground electrode under the caudal region of the tail.</li>
	<li>Evaluate the cochlear functions by measuring the ABR peak I (P1) amplitude. Automatically analyze the ABR waveforms with respect to the hearing thresholds and ABR P1 amplitude using the ABR peak analysis software as previously reported18.</li>
	<li>Calculate the ABR threshold shifts by subtracting the thresholds obtained before exposure. Compare the ABR threshold shifts in the three exposed groups to those of the non-exposed contralateral ears (control). Measure ABR amplitudes using ABR waveform during 80 dB SPL stimulation.</li>
</ol>
4. Histological assessment
NOTE: Histological assessment was performed as previously described14,15.
<ol>
	<li>HCs and cochlear synapse
	<ol>
		<li>Perform the pathological examination of the cochlea 1 month after LISW exposure.</li>
		<li>Confirm the depth of anesthesia via toe pinch before perfusion. After hemoperfusion with lactated Ringer&#39;s solution, perform transcardiac perfusion with 1 mL/g of 4% paraformaldehyde (PFA). After decapitation, remove the cochlea and perfuse directly with 4% PFA, followed by fixation at 4 &#176;C overnight.</li>
		<li>After fixation, decalcify the cochlea by shaking in 0.5 mol/L ethylenediaminetetraacetic acid (EDTA) solution for 2 days.</li>
		<li>Divide the demineralized cochlea into four pieces. After freezing each piece of cochlea on dry ice for 10 min, perform blocking at room temperature for 1 h in 5% normal horse serum simply conjugated with 0.3% Triton X for permeabilization.</li>
		<li>Use anti-myosin 7a (Myo7A), anti-C-terminal binding protein (CtBP2), and anti-neurofilament (NF) antibodies as primary antibodies and incubated at 37 &#176;C overnight. Use Myo7A, CtBP2, and NF antibodies to evaluate HCs, presynaptic ribbons, and cochlear nerve fibers, respectively.</li>
		<li>Wash off the unbound primary antibody with phosphate-buffered saline (PBS) for 5 x 3 min. Incubate the specimens with the appropriate secondary antibodies at 37 &#176;C for 2 h. After staining, wash the specimens for 3 x 5 min with PBS, and encapsulate the specimens on the slide glass with a water-soluble encapsulant using cover glass.</li>
		<li>For evaluation, acquire the entire image of the cochlea (divided into four pieces) at 10x magnification, and compute the cochlear frequency map using the referenced ImageJ software plug-in to precisely localize the specific cochlear regions at 12.0 kHz, 16.0 kHz, 20.0 kHz, and 24.0 kHz frequency.</li>
		<li>Calculate the rates of HC survival and the number of synapses at each frequency.
		<ol>
			<li>To calculate the rates of HC survival, count the numbers of surviving and missing HCs per 200 &#181;m length at each frequency, and calculate the survival rate of the HCs using equation (1) shown below: 
			HC survival rate (%) = (Number of surviving HCs / Number of surviving and missing HCs) &#215; 100&#160; (1)</li>
			<li>To calculate the number of synapses, obtain high-resolution z-stack images of the inner HC area using an oil immersion objective lens (63&#215;) with a 3.1x digital zoom and a 0.25 &#181;m step size under confocal fluorescence microscopy. Import the image stacks to ImageJ, and automatically count the CtBP2 puncta per IHC within a 50 &#181;m range in each image stack. Calculate the synaptic ribbons survival rate using equation (2): 
			&#8203;Synaptic ribbons survival rate (%) = (Number of synaptic ribbons in LISW exposed ears / Number of synaptic ribbons in control ears) &#215; 100&#160; (2)</li>
		</ol>
		</li>
		<li>For scanning electron microscopy (SEM), remove the cochlea, as previously described, and then fix with 2% PFA and 2.5% glutaraldehyde together at 4 &#176;C overnight. After decalcification of the cochlea by shaking in 0.5 mol/L EDTA solution at 4 &#176;C for 7 days, dissect the cochleae into four pieces for whole-mount preparation.</li>
		<li>Fix the tissues with 1% osmium tetroxide at 4 &#176;C for 30 min, dehydrate in 50% ethanol at room temperature for 10 min, repeat with 70 %, 80 %, 95 %, and then 100 % ethanol, sputter coating with osmium, and examine under an electron microscope at 5.0 kV, as previously reported14.</li>
		<li>Conduct a quantitative analysis of stereociliary bundle disruption in outer hair cells (OHCs) by calculating the ratio of disrupted stereocilia (number of disrupted OHC stereocilia/total number of OHC stereocilia) in each energy group using the SEM images14. Count the number of stereocilia per 100 &#181;m at the center of the 16.0 and 24.0 kHz regions. Designate one or more rows of OHC bundles that are bent toward the lateral side, tangled, or lacking their base as disrupted.</li>
	</ol>
	</li>
	<li>SGNs
	<ol>
		<li>To quantitatively assess the number of SGNs at 1 month after LISW exposure, perform transcardiac perfusion with 4% PBS, decapitate, remove the cochlea, and perform posterior fixation under the same conditions as described above. Decalcify the cochlea in 0.5 M EDTA for 1 week.</li>
		<li>After decalcification, immerse the cochleae in 30% sucrose overnight, embed them in the cryosectioning compound, freeze in liquid nitrogen to prepare sections near Rosenthal&#39;s canal at a thickness of 15 &#181;m, stain with hematoxylin and eosin, and view them under a light microscope14.</li>
		<li>For SGN density measurement, count the number of SGNs in the middle turn of the Rosenthal&#39;s canal and calculate SGN survival per control.</li>
	</ol>
	</li>
</ol>
5. Statistical analysis
<ol>
	<li>Perform statistical analyses using the software of choice.</li>
	<li>Analyze statistical differences in ABR threshold shift, HC, SGN, and synaptic counts using two-way repeated-measures ANOVA [&#34;frequency or cochlear parts (apical/middle/base)&#34; &#215; &#34;animal groups&#34;], two-way analysis of variance (ANOVA), followed by post-hoc Tukey&#39;s multiple comparison test.</li>
	<li>Present all data as mean &#177; standard error and set the statistical significance level at p &#60; 0.05.</li>
</ol>

Characterization of LISW Effects on Cochlear Pathophysiology in Mice

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The authors declare that they have no conflicts of interest.

This study aimed to validate a mouse model of blast-induced cochlear damage using LISW. Our findings demonstrated that following LISW application through the temporal bone, the exposed mice ear exhibited a consistent pathological and physiological decline in the cochlea, which was accompanied by an increase in LISW overpressure. These results indicate that this mouse model is appropriate for replicating various cochlear pathologies by adjusting the LISW output. Specifically, this LISW-induced cochlear dysfunction mouse m...

Hearing loss and tinnitus are among the most prevalent disabilities, reported in up to 62% of veterans1. Several blast-induced auditory complications, including sensorineural hearing loss (SNHL) and tympanic membrane perforation (TMP), have been reported in individuals exposed to blast overpressure2. Moreover, research on individuals exposed to blasts suggests that blast exposure frequently results in defects in auditory temporal resolution, even when the hearing thresholds are within normal range, which is known as &#34;hidden hearing loss (HHL)&#34;3. It is well established that there is a substantial loss of cochlear synapses between inner hair cells (IHCs) and auditory neurons (ANs) in blast-related cochlear pathology4. Synaptic degeneration results in impaired auditory processing and is a major contributing factor in the development of HHL5. Thus, auditory organs are fragile components containing complex and highly organized structures. However, the precise mechanism by which blast waves affect the inner ear at the cellular level remains unclear. This is because of the challenges in replicating the precise clinical and mechanical intricacies of blast injuries in laboratory settings and the complexity of blast-induced cochlear pathologies.
The primary component of a blast injury is the shock wave (SW), characterized by a rapid and high increase in peak pressure6. The complexity of blast injuries has been extensively investigated in numerous retrospective studies7,8,9. There are various devices for blast generation, such as compressed gas10, shock tubes11, and small-magnitude explosives12, at different levels of pressure. The pressure waveform of the SW generated by recently developed devices closely resembled that of an actual explosion. An important concept in establishing an animal model of blast-induced sensorineural hearing loss is to minimize concomitant injuries, other than auditory damage, to reduce animal death. Thus, blast injury studies have been developed in which shock tubes have been miniaturized and the output can be precisely controlled so that exposed animals rarely die. However, although these animal models usually develop complications, such as TMP, evaluation of cochlear function is difficult because of concurrent conductive hearing loss2. We previously performed an ear-protected animal study on blast injury using earplugs and found no incidence of TMP13. The earplugs could partially attenuate severe cochlear damage but not central auditory neurodegeneration or tinnitus development. Thus, earplugs protect the cochleae as well as the tympanic membrane. However, an animal model of blast-induced pure cochlear damage without TMP is required to study the cochlear pathophysiology caused by blast injuries.
We previously developed a topical blast injury model of the inner ear in rats and mice using a laser-induced shock wave (LISW)14,15. This method can be safely and easily performed at a standard laboratory level and has been used to generate models of lung and head blast injuries16,17. The energy of the LISW can be adjusted by changing the laser type and power, allowing control over the degree of cochlear damage. The LISW-induced cochlear injury model is valuable for studying the mechanisms of SNHL caused by blast injuries and investigating potential treatments. In this study, we describe detailed experimental protocols for creating a mouse model of blast-induced cochlear damage using LISW and demonstrate cochlear degeneration, including the loss of hair cells (HCs), cochlear synapses, and spiral ganglion neurons (SGNs), in an energy-dependent manner in mice following LISW exposure.

<table><tbody><tr><td>532 nm Q-switched Nd:YAG laser&amp;nbsp;</td><td>Quantel</td><td>Brilliant b</td><td></td></tr><tr><td>ABR peak analysis software</td><td>Mass Eye and Ear</td><td>N/A</td><td>EPL Cochlear Function Test Suite</td></tr><tr><td>Acrylic resin welding adhesive&amp;nbsp;</td><td>Acrysunday Co., Ltd</td><td>N/A</td><td></td></tr><tr><td>confocal fluorescence microscopy</td><td>Leica</td><td>TCS SP8</td><td></td></tr><tr><td>cryosectioning compound</td><td>Sakura</td><td>Tissue-Tek O.C.T</td><td></td></tr><tr><td>CtBP2 antibody</td><td>BD Transduction</td><td>#612044</td><td></td></tr><tr><td>Dielectric multilayer mirrors</td><td>SIGMAKOKI CO.,LTD</td><td>TFMHP-50C08-532</td><td>M1-M3</td></tr><tr><td>Digital oscilloscope</td><td>Tektronix</td><td>DPO4104B</td><td></td></tr><tr><td>Earphone</td><td>CUI</td><td>CDMG15008-03A</td><td></td></tr><tr><td>Hydrophone</td><td>RP acoustics e.K.</td><td>FOPH2000</td><td></td></tr><tr><td>Image J software plug-in</td><td>NIH</td><td>measurement line</td><td>https://myfiles.meei.harvard.edu/xythoswfs/webui/_xy-e693768_1-t_wC4oKeBD</td></tr><tr><td>Light microscope</td><td>Keyence Corporation</td><td>BZ-X700</td><td></td></tr><tr><td>Myosin 7A antibody</td><td>Proteus Biosciences</td><td>#25&amp;ndash;6790&amp;nbsp;</td><td></td></tr><tr><td>Neurofilament antibody</td><td>Sigma</td><td>#AB5539</td><td></td></tr><tr><td>Plano-convex lens</td><td>SIGMAKOKI CO.,LTD</td><td>SLSQ-30-200PM</td><td></td></tr><tr><td>Prism software</td><td>GraphPad</td><td>N/A</td><td>ver.8.2.1</td></tr><tr><td>Scanning electron microscope</td><td>JEOL Ltd</td><td>JSM-6340F</td><td></td></tr><tr><td>Small digital endoscope</td><td>AVS Co. Ltd</td><td>AE-C1</td><td></td></tr><tr><td>Ultrasonic jelly</td><td>Hitachi Aloka Medical</td><td>N/A</td><td></td></tr><tr><td>Variable attenuator</td><td>Showa Optronics Co.</td><td>N/A</td><td>Currenly avaiable successor: KYOCERA SOC Corporation, RWH-532HP II</td></tr><tr><td>Water-soluble encapsulant&amp;nbsp;</td><td>Dako</td><td>#S1964</td><td></td></tr></tbody></table>

research application of laser-induced shock wave for studying blast-induced cochlear injury

The ear is the organ most susceptible to explosion overpressure, and cochlear injuries frequently occur after blast exposure. Blast exposure can lead to sensorineural hearing loss (SNHL), which is an irreversible hearing loss that negatively affects the quality of life. Detailed blast-induced cochlear pathologies, such as the loss of hair cells, spiral ganglion neurons, cochlear synapses, and disruption of stereocilia, have been previously documented. However, determining cochlear sensorineural deterioration after a blast injury is challenging because animals exposed to blast overpressure usually experience tympanic membrane perforation (TMP), which causes concurrent conductive hearing loss. To evaluate pure sensorineural cochlear dysfunction, we developed an experimental animal model of blast-induced cochlear injury using a laser-induced shock wave. This method avoids TMP and concomitant systemic injuries and reproduces the functional decline in the SNHL component in an energy-dependent manner after LISW exposure. This animal model could be a platform for elucidating the pathological mechanisms and exploring potential treatments for blast-induced cochlear dysfunction.

LISW waveform 
The reproducibility of the LISW pressure waveform was measured 5x at 2.0 J/cm2 as follows. The waveforms were generally similar and stable and showed a sharp increase with time width, peak pressure, and impulse of 0.43&#177;0.4 &#181;s, 92.1 &#177; 6.8 MPa, and 14.1 &#177; 1.9 Pa&#8729;s (median &#177; SD), which corresponds to SW characteristics (Figure 1B). LISWs are characterized by a fast rise time, high peak pressure, short duration, and p...

Read this Scientific Article Protocol about Author Spotlight: Development of a Laser-Induced Shock Wave Animal Model Without Tympanic Membrane Perforation at JoVE.com

Author Spotlight: Development of a Laser-Induced Shock Wave Animal Model Without Tympanic Membrane Perforation

Here, we describe experimental protocols for creating an animal model of blast-induced cochlear injury using laser-induced shock wave (LISW). Exposure of the temporal bone to LISW allows the reproduction of blast-induced cochlear pathophysiology. This animal model could be a platform for elucidating cochlear pathology and exploring potential treatments for blast injuries.

Research Application of Laser-Induced Shock Wave for Studying Blast-Induced Cochlear Injury

The ear is the organ most susceptible to explosion overpressure, and cochlear injuries frequently occur after blast exposure. Blast exposure can lead to ...

research-application-laser-induced-shock-wave-for-studying-blast

Research

JoVE Journal

Medicine

517 Views.  National Defense Medical College. Om pure sensorische neuro-cochleaire disfunctie veroorzaakt door blootstelling aan blasten te evalueren, resulteert dit in tympanische membraanperforatie. We ontwikkelen een experimenteel diermodel van door ontploffing geïnduceerd cochleair letsel met behulp van een scheermes dat een schokgolf induceert. Deze methode kan trommelvliesperforatie en daarmee gepaard gaande systemische letsels voorkomen en de functionele achteruitgang van de sensorische neuro-geh...

Video: Auteur in de schijnwerpers: Ontwikkeling van een laser-geïnduceerd schokgolf diermodel zonder trommelvliesperforatie

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

Behavior

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Bioengineering

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

Neuroscience

Evaluating Primary Blast Effects In Vitro

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions,&#160;whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Evaluating Primary Blast Effects In Vitro

Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability.

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Low-intensity Blast Wave Model for Preclinical Assessment of Closed-head Mild Traumatic Brain Injury in Rodents

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of medical visits in the United States. There are currently no FDA-approved treatments available for TBI. The increased incidence of military-related, blast-induced TBI further accentuates the urgent need for effective TBI treatments. Therefore, new preclinical TBI animal models that recapitulate aspects of human blast-related TBI will greatly advance the research efforts into the neurobiological and pathophysiological processes underlying mild to moderate TBI as well as the development of novel therapeutic strategies for TBI.
Here we present a reliable, reproducible model for the investigation of the molecular, cellular, and behavioral effects of mild to moderate blast-induced TBI. We describe a step-by-step protocol for closed-head, blast-induced mild TBI in rodents using a bench-top setup consisting of a gas-driven shock tube equipped with piezoelectric pressure sensors to ensure consistent test conditions. The benefits of the setup that we have established are its relative low-cost, ease of installation, ease of use and high-throughput capacity. Further advantages of this non-invasive TBI model include the scalability of the blast peak overpressure and the generation of controlled reproducible outcomes. The reproducibility and relevance of this TBI model has been evaluated in a number of downstream applications, including neurobiological, neuropathological, neurophysiological and behavioral analyses, supporting the use of this model for the characterization of processes underlying the etiology of mild to moderate TBI.

We present here a protocol of a blast wave model for rodents to investigate neurobiological and pathophysiological effects of mild to moderate traumatic brain injury. We established a gas-driven, bench-top setup equipped with pressure sensors allowing for reliable and reproducible generation of blast-induced mild to moderate traumatic brain injury.

Traumatic brain injury (TBI) is a large-scale public health problem. Mild TBI is the most prevalent form of neurotrauma and accounts for a large number of ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Custom Overpressure Air System for Inducing CNS Injuries in Murine Models

System for Focal, Closed-System Central Nervous System Injury

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

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

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

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

Research Application of Laser-Induced Shock Wave for Studying Blast-Induced Cochlear Injury

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