JSM received funding from the United States Department of Veterans Affairs (VA) Office of Biomedical Laboratory Research &#38; Development (JSM, I01BX004896) and the VA Northwest Mental Illness Research Education and Clinical Center, a Congressionally-mandated VA entity investigating blast-induced brain injury and comorbid post-traumatic stress. JSM reports unrelated funding from the FY22 Traumatic Brain Injury and Psychological Health Research Program Translational Research Award (W81XWH-22-TBIPHRPTRA, Award Number HT94252310755). The authors thank Andrew Shutes-David for his editorial assistance.

All procedures were performed under protocol #1588223, approved by the Veterans Affairs Puget Sound Health Care System Institutional Animal Care and Use Committee and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
1. Animal care
NOTE: Animal models of LLB are limited solely by their availability and the capacity of the shocktube to accommodate their size. The described shocktube herein was designed specifically for use with mice.
<ol>
	<li>Use 3-4-month-old male or female C57BL/6J mice or other approved mouse strains/lines in accordance with experimental needs. Maintain the mice on a 12 h dark-light cycle in specific pathogen-free facilities with ad libitum access to food and water. Mice are typically socially housed with 4 or 5 in a cage. Maintain facility temperatures at 20-22 &#176;C.</li>
	<li>Bring cages containing blast and sham mice to a nearby holding area. Bring separate empty cages for transferring individual mice to and from the blast room.</li>
</ol>
2. Shocktube preparation
<ol>
	<li>(Safety check) Confirm that the necessary safety checks have been completed for the specific system. Ensure that the gas supply (helium) and master power are off/disconnected.</li>
	<li>Prepare membranes as needed for the specific number of low-intensity blasts to be conducted (Figure 1.1). Cut membrane dimensions as required for the specific shocktube used in this protocol:
	<ol>
		<li>Cut one sheet of plastic cling wrap into a 5.5&#34; x 5.5&#34; square to seal the spool, allowing it to pressurize.</li>
		<li>Cut one sheet of standard 8.5&#34; x 11&#34; copier paper (75 g/m2 weight) to 5.5&#34; x 11&#34;; fold the resulting sheet of paper in half to form a 5.5&#34; x 5.5&#34; square.</li>
		<li>Obtain one sheet of 500 G mylar membrane (125 &#181;m thickness). 
		NOTE: These sheets are not ruptured or significantly deformed by standard low-intensity blasting and can be reused for the duration of a day&#39;s procedure.</li>
	</ol>
	</li>
	<li>Take a square of cling wrap and a square of folded paper and set them out on a flat surface (Figure 1.2). Place the folded paper on top of the cling wrap and align the two with each other as best as possible (Figure 1.3). To expedite repetitive blasts, arrange all the membrane stacks now.</li>
	<li>Insert the mylar membrane between the driver and the spool by rolling it up into a small tube (about as big around as one&#39;s index finger; Figure 1.4,1.5). Insert it completely into the mechanism, and let go to allow it to unroll against the rubber seal that separates the driver section from the spool. Push the spool toward the driver to secure the mylar sheet in place; this will unseal the spool from the driven section of the shocktube.</li>
	<li>Place the fingers under the top half of the cling wrap and carefully roll both the cling wrap and paper toward you, ensuring they roll up together without becoming misaligned (Figure 1.6).</li>
	<li>Insert the membrane stack between the spool and the driven sections of the shocktube (Figure 1.7).</li>
	<li>Allow the membrane stack to unroll so that the plastic seal is facing toward the spool and the paper is facing toward the driven section of the tube (Figure 1.8). 
	NOTE: This orientation will create an airtight seal so that the system can be pressurized.</li>
	<li>Close the spool assembly (Figure 1.9,1.10). As appropriate, tighten the bolts by hand or hydraulically, securing the driver-spool-shocktube assembly so that the system can be pressurized. (Safety check;&#160;Figure 1.10) 
	NOTE: For hydraulic systems, ensure that the closure assembly&#39;s target pressure is reached to prevent misfires, which can require membrane replacement and slow the LLB exposure process. We use hydraulics to close our assembly at 500 psi.</li>
</ol>
3. Animal preparation
<ol>
	<li>Turn on the circulating water heating pad below the anesthesia chamber, with the temperature set to 37 &#176;C (Figure 1.11). Place an absorbent medical pad on top of the heat pad.</li>
	<li>In the holding room, remove one mouse from its home cage and place it into an empty transfer cage. Bring the caged mouse into the blast room.</li>
	<li>Turn the oxygen flow rate to 1.0 L/min (lpm) and the vacuum scavenge system on (Figure 1.12).</li>
	<li>Turn the isoflurane on to 5% (to induce rapid unconsciousness) and route the flow to the rodent anesthesia chamber (Figure 1.13).</li>
	<li>Place the mouse in the chamber to induce anesthesia (Figure 1.14).</li>
	<li>Once the mouse is fully anesthetized and displays stable breathing for an additional 30 s, reach into the chamber and ear punch the mouse for unambiguous long-term identification of the mouse throughout the remainder of the study. Doing this step now is necessary to avoid interfering with recovery times after the blast. Then, apply sterile ophthalmic lubricant to both eyes to prevent corneal drying.</li>
	<li>Remove the mouse from the chamber and place its nose into the nosecone (Figure 1.15). Switch the flow of anesthesia (e.g., isoflurane) from the induction chamber to the nosecone.</li>
	<li>Use small pieces of laboratory tape to lightly restrain the mouse&#39;s limbs against the gurney (Figure 1.16).</li>
	<li>After restraining the mouse, place a wire twist tie around each limb and twist tightly, securing the mouse to the gurney at the wrists and ankles (Figure 1.17). Place a larger twist tie around the chest, tying it very loosely such that the mouse&#39;s breathing is not restricted. This will serve as a secondary restraint mechanism in case any of the limb restraints come loose.</li>
	<li>Lift the mouse&#39;s tail and place it under the left foot to ensure it does not get pinched when the gurney is inserted into the shocktube (Figure 1.18).</li>
</ol>
4. LLB procedure
<ol>
	<li>Open the animal exposure section of the shocktube and orient the mouse so that it faces the oncoming blast wave (Figure 1.19).</li>
	<li>Secure/suspend the gurney in the animal exposure section (Figure 1.20).</li>
	<li>Close the door tightly for the animal exposure section, ensuring that the anesthetic flow tube is not pinched by the door (Figure 1.21).</li>
	<li>Reduce the anesthesia to 2.5-3% isoflurane, 1 lpm for the remainder of the session.</li>
	<li>Power the system as appropriate (Figure 1.22).</li>
	<li>Locate and connect the supply line for the compressed helium gas (Figure 1.23,1.24).</li>
	<li>Leave the blast room to access the blast tube control console in an adjoining room, and ensure no personnel or animals are left in the blast room. 
	NOTE: Hearing protection may be required by the institution or by operational conditions. Such conditions may include shocktube arrangements where the control console is located in the same open space as the shocktube.</li>
	<li>From the console, turn on the acquisition software to record the blast event (see the green box in Figure 1.25). 
	NOTE: For these procedures, we collect sensor data at a 20 kilo hertz (kHz) sampling rate, which is then processed using LabView software. We recommend acquiring sensor sampling at &#8805;10 kHz to achieve high-quality time versus pressure curves.</li>
	<li>Disengage any safety lock (e.g., power control keys, which are depicted by a green arrow in Figure 1.26).</li>
	<li>Close both gas vents and passively pressurize the spool (Figure 1.27). Do not use the driver side. Continue to fill until the membrane ruptures on its own at the target peak psi as determined by the number of membrane sheets used.</li>
	<li>Record the peak pressure, positive phase duration, and impulse at the animal location. (Figure 1.28). Turn off the fill mechanism.</li>
	<li>Return to the shocktube, disconnect the helium feed line, and turn off the power supply to the blast control circuit (Figure 1.29).</li>
	<li>To conduct repeated LLB exposures on the same animal, open the spool, remove the spool membrane stack, and then roll and insert another spool membrane stack (Figure 1.30, 1.31, 1.32). Flatten the membrane stack and reclose the assembly. 
	NOTE: To model the clinical experience of low-level blast exposures during empirically defined SOF training, we expose mice to 5-6 LLBs per day, capping daily exposures to a conservative ~20 cumulative total psi45. Studies emphasizing mechanistic and dose-response relationships may alternatively choose to use a consistent number of LLB exposures with defined overpressure parameters per session.</li>
	<li>After the final LLB for the current animal, remove it from the shocktube, leaving the anesthesia on (Figure 1.33).</li>
	<li>Untie the animal while it is under anesthesia. Remove it from the anesthesia nosecone, placing it on its back onto the heated water pad (Figure 1.34).</li>
	<li>Once the animal has been placed on the water pad, start a timer and record the amount of time until the mouse flips over onto its ventral side (i.e., its stomach) on its own (Figure 1.35). Record this time as the righting time. Once the mouse recovers, return it to the home cage and continue monitoring as needed.</li>
</ol>
5. Multiday procedures
<ol>
	<li>To model routine LLB exposures from breaching charges used during SOF Close Quarter Battle training, perform repeated daily exposures on the mice 5 days a week (Monday through Friday) for a total of 15 days across 3 standard work weeks.</li>
</ol>
6. Altering peak LLB pressures
<ol>
	<li>Increase peak pressure through the use of stronger membrane materials or by simply stacking additional membranes. For example, use Mylar Roll Clear 0.005 (500 G) membrane to produce ~20 psi peak pressure (when used as both driver and spool membranes) or Mylar Roll Clear 0.002 (200 G) membrane to produce ~10 psi peak pressure.</li>
	<li>Adjust the parameters for the positive phase duration and impulse of the blast to meet experimental needs. To adjust positive phase durations and impulses, empirically determine target conditions by substituting compressed gas sources47,49 or changing the driver length whenever possible. The above protocol uses helium to create a sharp peak pressure and waveform similar to an idealized Friedlander curve.</li>
</ol>
7. Tissue collection
NOTE: Tissue collection practices can be adjusted according to experimental needs.
<ol>
	<li>Anesthetize the mouse via intraperitoneal injection with 210 mg/kg of pentobarbital.</li>
	<li>Place the mouse into a mouse or rat cage with bars or a premade mesh; place the caged mouse into a fume hood.</li>
	<li>Once the mouse is unresponsive, place it on its back on the bars on top of the cage and close its mouth around one of the bars to help it stay in place during perfusion.</li>
	<li>Grab the skin of the stomach, pull it upwards, and use a pair of large scissors to cut a hole in the abdominal cavity, being careful not to cut any of the organs. Continue to cut farther down along the base of the ribs to allow for freer articulation of the ribcage.</li>
	<li>Using a hemostat, approach the mouse from the side and grab the tissue directly on top of the ribcage, rolling the hemostat back to keep the base of the ribcage angled in an easily accessible position. Use a pair of forceps or a similar tool to hold the hemostat in place.</li>
	<li>Using a small pair of surgical scissors, carefully cut the diaphragm to allow access to the heart. Use a pair of forceps to gently angle the heart such that the bottom is facing directly out of the open base of the ribcage. Work quickly so the heart will still be beating during perfusion.</li>
	<li>If collecting blood, hold the heart with a pair of forceps and carefully pierce the right ventricle using a 3 mL syringe tipped with an 0.5&#34; 25 G needle. Insert from the bottom of the ventricle and go in lengthwise, being careful not to pierce the opposite side of the ventricle. Gently pull on the syringe until 0.5-1.0 mL of blood has been collected or the flow stops, and then remove the syringe.</li>
	<li>Use a pair of surgical scissors to cut a small incision in the right atrium to allow blood and perfusate to drain. Hold the heart with a pair of forceps, and carefully insert a 25 G butterfly needle into the left ventricle, inserting from the bottom. Hold the butterfly needle in place with a holding clamp or by hand.</li>
	<li>Perfuse the animal.
	<ol>
		<li>Connect a syringe containing 50 mL of phosphate-buffered saline (PBS) to a butterfly needle and perfuse at a rate of approximately 10 mL/min. Look for blanching of the liver as a sign of proper perfusion. After the syringe is emptied, disconnect it from the butterfly needle.</li>
		<li>For preparation of tissues for microscopy, replace the empty PBS syringe with a syringe containing 50 mL of 10% neutral buffered formalin (NBF) or 4% formaldehyde solution. Repeat the above steps to perfuse with formalin. 
		NOTE: The perfused mouse should be observed to twitch during perfusion; this should result in full body rigor or rigidity after the procedure is completed.</li>
	</ol>
	</li>
	<li>Remove the butterfly needle from the heart and remove the mouse from the cage bars for tissue collection.</li>
	<li>Remove and subdissect the target organs according to need; be careful to perform procedures on ice when fresh, unfixed materials are collected.</li>
	<li>Flash freeze any unfixed tissues that were collected in liquid nitrogen and store them at -80 &#176;C until used in protocols assaying protein or RNA targets.</li>
	<li>For fixed tissues, remove to a labeled 50 mL conical tube filled with formalin (one tube per organ).</li>
</ol>

Shocktube Method to Model Highly Repetitive Low-Level Blast in Mice

The authors have no financial interests to disclose.

We cannot adequately treat what we inadequately understand, and we do not yet understand the injury mechanisms related to highly repetitive LLB exposure. Many SOF personnel report the development of health-related impairments thought to be related to highly repetitive LLB exposure within five to ten years of operational service50,51. Some SOF personnel develop acute traumatic brain injury (TBI)-like neurocognitive effects immediately after LLB exposure39. Furthermore, clinicians report that symptoms resulting from blast exposure are frequently refractory to traditional treatments, which may drive SOF and clinicians to look to alternate treatments52,53. Despite the frequent exposure of SOF to LLB and overpressure mechanisms45, the severity and treatment resistance of the resulting symptoms, and the documented pattern of blast-related astroglial scarring51, the long-term health outcomes remain relatively unknown. Clinicians and military leadership rely on modeling research to uncover injury mechanisms and pathophysiology. These models are critical to developing policies and strategies to identify, interrupt, prevent, and treat the pathology process early.
Crucially, mouse modeling of common military LLB exposures is expected to inform health-prediction models. Clinical practice would benefit from LLB predictive models that identify who may be at greatest risk for blast-related pathology, which blast properties provoke the most serious outcomes, and how the disease process may evolve based on the chronicity, dosing, or specificity of the blast exposure. Thus, modeling repetitive LLB exposure is essential in developing hypotheses and predictions for how exposures will impact the health outcomes of SOF and other Service members. Prediction and injury mechanism models would inform diagnostics and treatment, as well as return-to-duty decisions based on symptoms and exposure.
The study of blast-induced TBI (bTBI) in mice has seen significant advancements in recent years, particularly with the development of models that predict outcomes following chronic repetitive mild bTBI in humans54,55. Whereas the study of mid-to-high level blast exposure using shocktubes is well developed with hundreds of PubMed indexed articles46,56,57,58, shocktube use in studies of blasts near routine military training overpressures (&#60;6 psi peak pressure40) is less developed, with fewer than ten articles identified in a recent PubMed search19,20,22,23,26,27,28. To facilitate the development of this understudied field, the presented model focuses on key considerations for consistent LLB overpressures in mice, post blast recovery, and monitoring while noting several distinct advantages of this model over the use of open-field explosives. Indeed, we argue that the described laboratory LLB model may enable the development of predictive models of clinical outcomes following chronic repetitive LLB.
The LLB model offers critical advantages over open-field explosive blast models, particularly in terms of animal welfare. Open-field models may result in 3-8% mortality rates25,36, whereas this lab-based LLB model shows no loss. This distinction is crucial, especially when simulating the high cumulative exposures typical of military training, where virtually no trainees experience fatal outcomes from LLB exposure. The apparent absence of apnea or other causes of death, such as lethal pulmonary trauma, ensures the model&#39;s reliability and consistency, positioning it as a preferred choice for studies on the clinically relevant effects of repetitive LLB.
This protocol is specific for an &#34;open-ended&#34; shocktube with a three-part design, consisting of driver, spool, and driven sections. Highly repetitive LLBs may be achievable with other shocktube designs with appropriate modifications to the protocol. Open-ended shocktube designs are frequently used for the study of blast-induced neurotrauma46,47,48. The open-ended shock tube, featuring an open exit end, allows the generated shock wave to propagate freely down the length of the tube where it encounters its target (e.g., the animal subject) before exiting the opposite end of the tube. This design facilitates the reproduction and study of relatively pure primary blast overpressures approximating the properties of blast explosions as they would occur in the open field48. As a result, the fidelity of the empirically measured blast overpressure wave is compared with an idealized Friedlander wave; this allows the evaluation of the tube performance to produce a specific overpressure event. To model LLB exposure, we use a previously described48, custom-built, open-ended blast tube originally designed to reproduce the effects of HLB detonations of over 200+ lbs of trinitrotoluene (TNT) at a standoff distance of ~25 feet. To enable high peak overpressures, a gas is pressurized into the driver, which is separated from the spool by a membrane, sealing the gas in the driver. The spool, in turn, is also separated from the open-ended section by another membrane. This second membrane allows the spool to be separately pressurized. The dual chamber system allows the gases in the driver to be pressurized past the membrane&#39;s normal point of rupture. This occurs because the pressurized spool acts as a buffer, supporting the membrane at the interface of the driver and the spool, thus preventing its rupture. When the shock tube operator desires to generate a shockwave at the target pressure, an electronic valve vents gas from the spool, rapidly dropping pressure in the spool and allowing the overpressurized gas in the driver section to rupture both the driver and spool membranes and rapidly expand down the length of the tube where it encounters the animal in the target zone. The key modification enabling the study of LLB in high-performance tubes of this design is that we block off the driver and only use the spool in combination with low-threshold membranes.
To ensure the reliability and reproducibility of LLB experiments, certain actions must be taken during the setup. Tightly securing the arms and legs at the wrists and ankles is crucial. This minimizes variability in bodily movement and blast exposure and prevents unintended injuries that could confound results. Additionally, rotating wrists and ankles inward helps direct the movement of the appendages toward the animal midline, reducing the risk of distal injuries that might affect subsequent motor performance assessments. The straightening of the head and spinal curvature is another essential factor in ensuring uniform blast exposure across subjects, as it helps to reduce potential differences in range of motion. Increasing the percentage of isoflurane that is used for anesthesia is recommended for protocols spanning multiple days or weeks. This adjustment helps maintain consistent anesthesia depth throughout the extended experimental duration. In our experience, an increase in 0.5% isoflurane is sufficient to maintain adequate anesthesia.
However, anesthesia delivery via nosecone may not be possible for all blast tube designs, especially for those with full enclosures that do not allow the insertion of the tubing into the driven section. In such events, injectable anesthetics may be preferable. We recommend determining how much time is required for the delivery of the repeated sequential blasts and then administering sufficient anesthetic to maintain unconsciousness throughout the procedure. Additional animal welfare checks may be necessary during the development of this modified method to ensure proper anesthesia maintenance. Furthermore, the use of injectables may render postacute response monitoring, such as the collection of righting time measures, impossible.
Ethical considerations are paramount in animal research, and this lab-based LLB model incorporates comprehensive post-blast recovery and monitoring protocols. Humane endpoints after blast exposure, including difficulty breathing, inability to right themselves, non-ambulatory status after a 2 h observation period, seizure-like movements, awkward movements, vision impairment, and evidence of internal bleeding or fractured limbs, are closely observed. Notably, LLB blast mice have not exhibited any of these conditions in our experiments. However, limb fractures can occur during HLBs, often due to operator error. Mitigating this risk involves rotating the hands and feet toward the animal midline during gurney securing. This technique prevents the blast wind from sweeping the appendages backward and breaking the associated bones.
This repetitive LLB model&#39;s advantages extend beyond ethical considerations to practical and methodological aspects. Its lab-based design eliminates the need for handling explosives, thereby enhancing safety and accessibility. The model is highly reproducible and customizable, allowing researchers to influence exposure parameters through the use of different gas types, device settings, and membrane strengths. Helium, which is chosen here for its ability to reproduce open-field explosion kinetics49, may provide a reliable baseline47,59,60. Adjusting peak pressure is achieved empirically by modifying the retention membrane thickness or strength, enabling fine-tuning for specific experimental requirements. Lastly, the LLB model eliminates the impact of seasonal or weather variations on data, animal exposure, and other experimental factors. This consistency ensures robust and reliable results, making this repetitive LLB model an invaluable tool for longitudinal and highly repetitive blast research.
Understanding blast-related neurotrauma requires elucidating injury mechanisms, blast intensity metrics, and threshold values. However, uncertainties surround human brain injury mechanisms in blast scenarios. Previously proposed criteria for human injury following blast exposure have relied on animal studies, yet it is challenging to directly apply these studies to humans due to incomplete scaling criteria across species61. Lung injury scaling based on animal body mass is an exception, given the presence of accepted criteria62,63. Proposed scaling laws for brain effects, based on body64,65 or brain mass66, however, overlook known and unknown anatomical differences, especially concerning the protective structures inside and around the brain. Mass scaling predicts higher injury risks in smaller-bodied species, which are contradicted by studies both in birds67,68,69 and humans70. Developing accurate scaling laws therefore demands an empirical understanding of the relationship between external blast event intensity and internal brain effects across species. In the case of LLBs, very little is known about either single or chronic exposure in either animal models or people. As a result, the empirical studies needed to inform the development of future scaling laws at the LLB intensity range may be catalyzed by our method.
In summary, this laboratory-based shocktube model represents a significant advancement in the study of the chronic effects of LLB exposure in mice. By incorporating procedures for modeling consistent overpressures, prioritizing post-blast recovery and monitoring, and highlighting distinct advantages over alternative models, this lab-based LLB model may provide a reliable and ethical choice for advancing our understanding of injuries related to chronic LLB exposure.

Low-level blast (LLB) exposure occurs when individuals or structures experience relatively low magnitudes of explosive force, typically arising from small industrial accidents, controlled demolitions, or certain military training activities. In contrast, high-level blast (HLB) exposure entails exposure to intense and potentially destructive magnitudes of explosive force, commonly encountered in military combat, terrorist attacks, or large-scale accidental explosions. The primary distinction between LLB and HLB therefore lies in the intensity of the explosive events and, by extension, the ability of exposed persons to tolerate repeated exposures before experiencing physical or functional injury. In this regard, the effects of HLB exposure tend to be more obvious than the effects of LLB exposure. Because of this, persons with significant LLB exposure may be at increased risk for slowly developing injuries or deficits that go undetected until their cumulative effects become discernable.
Ongoing research aims to enhance our understanding of how the properties of blast exposure, such as intensity or repetition, may cause injury so that we can better guide prevention and medical management. In military medicine, understanding the clinical implications of blast exposure is of paramount importance, and as a result, animal models capable of informing those outcomes are needed. Although animal models have helped elucidate the effects of HLB, the effects of LLB exposures remain largely understudied. Numerous modeling studies examine the effects of blast overpressures near or above 10 pounds per square inch (psi) peak pressure1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18, but few reports focus on pressure levels ranging from 1 to 7 psi19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36, which are more common in military training environments37,38,39,40 and fall near the historical threshold of 4 psi for safe environmental exposure. Thus, broader dissemination of methods for the study of frequently used peak pressures of LLB may help catalyze rapid clinical insights for application to military medicine and force optimization.
A significant association between the occupational risk of LLB and diverse clinical diagnoses is emerging from epidemiological investigations of military LLB41,42,43,44. These studies support a poorly defined dose-dependent relationship, with repetitive LLB exposures demonstrating heightened risks41. This suggests that increasing cumulative blast exposure plays a crucial role in shaping clinical outcomes in military settings.
Previous animal modeling studies of LLB under 10 psi have primarily used explosives or shocktube systems to investigate the effects of exposure. Although these models typically examine the effects of one to three exposures, they have nonetheless contributed to a growing understanding of the mechanistic19,20,30,31, neuropathological29,31,33, and behavioral consequences19,20,23,25,32,34, associated with low-intensity blast exposures that are typical of the military training environment.
Studies examining single LLBs generated by open-field explosives have reported evidence of subtle brain pathologies and behavioral changes frequently associated with posttraumatic stress. Woods and colleagues24 were unable to detect microscopic brain injury at 2.5-5.5 psi, but they did detect quantitative changes in brain tissue glycosphingolipids by mass spectrometry. Using the same peak pressures and experimental design, Rubovitch and colleagues25 observed behavioral changes following blasts that occurred with a similar lack of brain pathology when measured by light microscopy. However, in subsequent pathological investigation, unambiguous ultrastructural damage to brain myelin, mitochondria, neurons, and neurovasculature was identified by electron microscopy29,30,31,32,33 in 6.7 psi LLB-exposed mice. Interestingly, several LLB studies using open-field explosives with pressures of ~10 psi and less report approximately 3-8% mortality after a single exposure25,36.
Similar results have been previously noted by several studies using laboratory shocktubes. In studies examining single LLBs produced by shocktubes, evidence has been found of neural cytoskeletal injury and changes in neuronal firing patterns developed after exposure to a single 1.7 psi blast22. At 4 psi, corpus callosum dysfunction was reported to accompany neurobehavioral deficits in LLB-exposed rats23. Compared to the blast duration measured in air, Chavko and colleagues27 found that the positive phase duration of the blast overpressure was significantly longer in the brains of rats exposed to 5.8 psi. Biosignatures of similar injury responses may be supported by a study in mice following 7.5 psi exposure in which Ahmed and colleagues35 report detectable changes in serum levels of specific inflammatory, metabolic, vascular, and neural injury proteins up to a month after exposure. Interestingly, this study also reported 4.5% mortality at 24 h following exposure.
In studies examining three shocktube LLBs over a single 20 min exposure session, LLBs between 1.4 and 8.7 psi caused psi-dependent increases in intracranial pressure (ICP) in rats, with observable ICP changes taking longer for lower psi20 and resulting in cognitive changes19,20. Using pigs, the same group determined that three 4 psi LLB exposures from a variety of military equipment were sufficient to cause histological neuropathology when the animals were placed in gunner positions simulating human use of the equipment21.
These studies collectively illustrate the diverse effects of LLB exposure that may occur under conditions of limited exposure and recovery periods. Repetitive LLB exposure appears to induce persistent cognitive and behavioral deficits, emphasizing the need for a nuanced understanding of the cumulative effects so we can better determine when those effects may become clinically significant; this is particularly relevant for military trainees who are exposed to high levels of repetitive LLB. To achieve this, new studies are required since the current literature does not adequately model the clinical experiences of routine military training exposures that exceed one to a few blasts over the course of a few days.
Special Operations Forces (SOF) may endure significant and highly repetitive LLB during routine exposures. A recent study estimates the representative exposure anonymized across all positions in an explosive entry breaching team to be as high as 184 cumulative peak psi over the course of one training week42. This is based, in part, on a conservative estimate of 6 breaching charges used per day, with an average of 4 psi peak pressure each, as measured by personnel-mounted blast gauges; it does not account for flashbangs and other devices45. A routine training cycle may last several weeks. To facilitate the study of clinical LLB experiences, such as those of training SOF members, we present a laboratory-shocktube model of highly repetitive LLB exposure. The method, based on established pneumatic shocktube systems46,47,48, allows for highly reproducible investigations of pressures of 2 psi and higher. The procedure is not dependent on external factors such as weather, results in no observed mortality, and is lab-based. As a result, the method enables sustained, daily repetitive LLB exposures in the same subjects for studies lasting weeks to months, facilitating the high-fidelity investigation of military training.

<table><tbody><tr><td>Adroit Thermal Recirculating Heat Pump (120 V)</td><td>Parkland Scientific</td><td>HTP-1500</td><td></td></tr><tr><td>Copy paper, 75 g/m&lt;sup&gt;2&lt;/sup&gt; weight</td><td>Staples</td><td>897804</td><td></td></tr><tr><td>Disposable Absorbant Blue Pads</td><td>VWR</td><td>82020-845</td><td></td></tr><tr><td>Forane Inhalant Solution</td><td>MedLine</td><td>10019-360-60</td><td></td></tr><tr><td>Helium</td><td>Linde</td><td>UN1046</td><td></td></tr><tr><td>Laboratory tape (1&quot;)</td><td>VWR</td><td>89098-076</td><td></td></tr><tr><td>LabView software</td><td>Emerson</td><td>V 2011</td><td></td></tr><tr><td>Medical oxygen</td><td>Central Welding Supply</td><td>UN1072</td><td></td></tr><tr><td>Mylar, 0.005 thickness</td><td>Tapp Plastics</td><td>22934</td><td></td></tr><tr><td>Plastic cling wrap</td><td>Santa Cruz Biotechnology</td><td>sc-3687</td><td></td></tr><tr><td>Plastic twist ties&amp;nbsp;</td><td>VWR</td><td>11215-940</td><td></td></tr><tr><td>Pneumatic Shocktube (with driver and spool sections; target area sized for mice, 20 kHz sampling rate pressure sensors, control and acquisition software)</td><td>BakerRisk, San Antonio, TX</td><td>custom</td><td></td></tr><tr><td>Reusable Heavy Duty Heating Pad (12&quot; x 18&quot;)</td><td>Parkland Scientific</td><td>121218</td><td></td></tr><tr><td>Scissor-style, Rodent Ear Punch</td><td>Kent Scientific</td><td>INS750076-2</td><td></td></tr><tr><td>Sliding Top Chambers for Traditional Vaporizers</td><td>Kent Scientific</td><td>VetFlo-0530SM</td><td></td></tr><tr><td>VetFlo Isoflurane Vaporizer</td><td>Kent Scientific</td><td>VetFlo-1210S</td><td></td></tr></tbody></table>

modeling highly repetitive low-level blast exposure in mice

Exposure to explosive blasts is a significant risk factor for brain trauma among exposed persons. Although the effects of large blasts on the brain are well understood, the effects of smaller blasts such as those that occur during military training are less understood. This small, low-level blast exposure also varies highly according to military occupation and training tempo, with some units experiencing few exposures over the course of several years whereas others experience hundreds within a few weeks. Animal models are an important tool in identifying both the injury mechanisms and long-term clinical health risks following low-level blast exposure. Models capable of recapitulating this wide range of exposures are necessary to inform acute and chronic injury outcomes across these disparate risk profiles.
Although outcomes following a few low-level blast exposures are easily modeled for mechanistic study, chronic exposures that occur over a career may be better modeled by blast injury paradigms with repeated exposures that occur frequently over weeks and months. Shown here are methods for modeling highly repetitive low-level blast exposure in mice. The procedures are based on established and widely used pneumatic shocktube models of open-field blast exposure that can be scaled to adjust the overpressure parameters and the number or interval of the exposures. These methods can then be used to either enable mechanistic investigations or recapitulate the routine blast exposures of clinical groups under study.

While investigating experimental outcomes in mice following exposures to explosive blast forces, recording and characterizing the event through pressure versus time analysis is crucial for evaluating the success of the experiment. This method, which involves measuring the dynamic changes in pressure during the blast, helps investigators understand the effects of blasts on biological systems.
In successful experiments, pressure recordings exhibit a well-defined and controlled wave pattern. The pressure rise is sharp, reaching peak values within expected times (Figure 2). The subsequent pressure decline follows a predictable curve, exemplified by the Friedlander waveform, indicating efficient dissipation of energy. In terms of injury assessment, no overt signs of injury are present in LLB experiments, even when conducting highly repetitive LLB exposure with up to six blasts occurring within 15-20 min (Figure 3). However, an analysis of righting times following repetitive LLB exposure indicates that blast mice return to consciousness faster than sham mice (Figure 4). Thus, repetitive LLB results in reproducible changes in acute neurobehavioral arousal responses after exposure.
Suboptimal experiments may display irregular pressure profiles. Instances in which peak pressures are unexpectedly depressed may indicate a premature or slow release of gas, preventing the sharp release of gas expansion down the length of the driven shocktube section to encounter the animal in the target area. Premature loss of gas pressure is often the result of improperly sealed driver or spool sections. This can result from flaws in the membrane or inadequate tightening of the driver-spool-shocktube assembly. In such cases, biological samples may exhibit reduced signs of trauma.
Data interpretation involves linking pressure-time profiles with observed biological responses. Successful experiments demonstrate that the chosen blast parameters, such as peak pressure and duration, elicit the expected or established biological responses under investigation. Correlations between specific pressure features and biological outcomes aid in establishing causal relationships. Longitudinal studies are enabled by this protocol due to the lack of observed animal loss for study time points as long as 6 months after the final LLB (Figure 5).
The range of clinical outcomes following LLB exposure is subtle and poorly understood. Repetitive exposure to LLBs has historically been considered subinjurious for both people and mice. This is supported by a quick return to normal ambulation, behavior, and physical activity following exposures at 2-5 psi. However, the lack of overwhelming acute neurosensory symptoms or behavioral changes does not preclude the existence of negative insidious effects. Because LLB-related phenotypes are subtle at best, the full range of effects is an area of active investigation and may require considerable time or repetition to provoke clinically significant outcomes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66592/66592fig01.jpg" /> 
Figure 1: Procedural steps for the shocktube model of repeated murine LLB. Following both the preparation of the shocktube (Steps 1-10) and the animal preparation stages (Steps 11-18), mice are exposed to one or more LLBs (Steps 19-32), before being removed from the tube (Step 33). Mice are then placed on their backside onto a warmed heating pad (Step 34). The amount of time it takes the animal to flip over onto their ventral side is recorded as the righting time (Step 35). Abbreviation: LLB = Low-level blast. <a href="https://www.jove.com/files/ftp_upload/66592/66592fig01large.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/66592/66592fig02.jpg" /> 
Figure 2: Representative pressure-time curves for exposures near 4 psi. (A) Additive stacks provide linear peak pressures across the range of 2-4.5 peak psi. Representative pressure versus time (milliseconds) profiles averaged from 3-6 shocktube blasts (red) as compared to the idealized Friedlander curves (blue) for (B) 1 sheet, (C) 2 sheets, (D) 3 sheets, and (E) 4 sheets. <a href="https://www.jove.com/files/ftp_upload/66592/66592fig02large.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/66592/66592fig03.jpg" /> 
Figure 3: Intersubject Interval. Set up and execution of a single blast requires on average 9.8 &#177; 1.9 min (mean &#177; standard error of the mean (sem)). Additional blast exposures require an additional 1.7 &#177; 0.4 min per event (mean &#177; sem). Dots represent results from individual animals. <a href="https://www.jove.com/files/ftp_upload/66592/66592fig03large.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/66592/66592fig04.jpg" /> 
Figure 4: Daily righting times during 3 weeks of highly repetitive LLB exposures. The graph represents the sham-normalized righting times over 3 weeks of LLB exposure. LLB mice were subject to 6 daily blast exposures for a total of 90 total LLB exposures occurring over 15 days. Mean overpressure characteristics were (&#177; sem) 3.05 &#177; 0.07 peak psi, 0.94 &#177; 0.04 positive phase duration, and 2 &#177; 0.1 psi * msec impulse. p-values reflect results from 2-way ANOVA. Abbreviation: LLB = Low-level blast. <a href="https://www.jove.com/files/ftp_upload/66592/66592fig04large.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/66592/66592fig05.jpg" /> 
Figure 5: Effects of the laboratory shocktube LLB model on animal attrition following highly repetitive LLB exposures. Attrition rates for sham (N = 24) and LLB mice (N = 32) from the first LLB exposure (day 1) through all study exposures (ending day 19) and following a 6-month recovery period (day 199). There was no significant difference between the attrition rates of sham and LLB groups over the observed period. LLB mice experienced an average of 62 exposures at an average of 4.78 &#177; 0.01 peak psi and 3.16 &#177; 0.023 psi&#8729;ms impulse. Exposures were administered to mice 5 days per week (i.e., Monday-Friday) for 3 consecutive weeks to model recently reported SOF overpressure exposures during routine breaching training45. Abbreviation: LLB = Low-level blast; SOF = Special Operations Forces. <a href="https://www.jove.com/files/ftp_upload/66592/66592fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Deciphering the Long-Term Effects of Low-Level Blast Exposures in Mice

Presented here are methods for producing repeated low-intensity blast exposures using mice.

Modeling Highly Repetitive Low-level Blast Exposure in Mice

Exposure to explosive blasts is a significant risk factor for brain trauma among exposed persons. Although the effects of large blasts on the brain are ...

modeling-highly-repetitive-low-level-blast-exposure-in-mice

Research

JoVE Journal

Neuroscience

897 Views.  United States Army Special Operations Command. Presented here are methods for producing repeated low-intensity blast exposures using mice.

Video: Author Spotlight: Deciphering the Long-Term Effects of Low-Level Blast Exposures in Mice

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Cancer Research

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Establishment of a Robust and Reproducible Model of Radiation-Induced Skin and Muscle Fibrosis

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. Radioactive beams can penetrate through the skin and affect the structures in their path including skin, muscles, and internal organs. Skin is the first structure to get exposed to radiation and is susceptible to develop chronic fibrosis, which is challenging to treat. Currently, limited treatment options show moderate efficacy in mitigating radiation-related skin fibrosis. A key factor hindering the development of effective countermeasures is the absence of a convenient and robust model that could allow for translation of the experimental findings to humans. Here, a robust and reproducible murine hind limb skin fibrosis model has been established for prophylactic and therapeutic evaluation of possible agents for functional and molecular recovery.
The right hind limb was irradiated using a single dose of 40 (Gray) Gy to induce skin fibrosis. Subjects developed edema and dermatitis in the early stages proceeded by visible skin constriction. Irradiated limbs showed a significantly reduced limb range of motion in the following weeks. In late stages, acute side effects subsided, yet chronic fibrosis persisted. A gait index was performed as an additional functional assay, which demonstrated the development of functional impairment. These non-invasive methods demonstrated reliable measurements for tracing fibrosis progression, which is supported by histological analyses. The radiation dose, application, and post-irradiation analyses employed in this model offer a vigorous and reproducible method for studying radiation-induced skin fibrosis and testing the efficacy of therapeutical agents.

Here we present a protocol to induce radiation-induced skin fibrosis in the hind limb of mice and perform post-irradiation measurements of chronic impairment via limb excursion and gait index analyses to evaluate the functional outcome. The model elucidates radiation-related skin fibrosis mechanisms and is useful in subclinical therapeutic studies.

Radiation-induced skin fibrosis (RISF) can result from a plethora of scenarios including cancer therapy, accidental exposure, or acts of terrorism. ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Medicine

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

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 ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

Biology

A Mouse Model of Pulmonary Fibrosis Induced by Nasal Bleomycin Nebulization

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse models continue to be an important tool for developing new anti-fibrotic strategies. In this study, intrapulmonary administration of bleomycin (BLM) is carried out by nasal nebulization to create a mouse model of pulmonary fibrosis that closely mimics clinical disease characteristics. C57BL/6 mice received BLM (7 U/mL, 30 min/day) by nasal nebulization for 3 consecutive days and were sacrificed on day 9, 16, or 23 to observe inflammatory and fibrotic changes in lung tissue. Nasal aerosolized BLM directly targeted the lungs, resulting in widespread and uniform lung inflammation and fibrosis. Thus, we successfully generated an experimental mouse model of typical human pulmonary fibrosis. This method could easily be used to study the effects of the administration of various nasal aerosols on lung pathophysiology and validate new anti-inflammatory and anti-fibrotic treatments.

Various animal models of pulmonary fibrosis have been established using bleomycin to clarify the pathogenesis of pulmonary fibrosis and find new drug targets. However, most pulmonary fibrosis models targeting lung tissue have uneven drug administration. Here, we propose a model of uniform pulmonary fibrosis induced by nasal bleomycin nebulization.

Pulmonary fibrosis is characteristic of several human lung diseases that arise from various causes. Given that treatment options are fairly limited, mouse ...

Modeling Highly Repetitive Low-level Blast Exposure in Mice

Summary

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