This work was supported by the Key Social&#160;Development Foundation of&#160;Jinhua&#160;Municipality&#160;(No. 2020-3-071), Zhejiang College Student&#160;Innovation and&#160; Entrepreneurship Training&#160;Program (No: S202310345087,&#160;S202310345088) and Zhejiang Provincial&#160;College Students&#39;&#160;Science and Technology&#160;Innovation Activity Plan Project (2023R404044). The authors thank Miss Emma Ouyang (first-year student of Johns Hopkins University, Bachelor of Science, Baltimore, USA) for language editing the article.

All procedures involved in this protocol were performed under the Institutional Animal Care and Use Committee approval (Zhejiang Normal University, Permit Number, dw2019005) and in compliance with the ARRIVE and the NIH Guide for the Care and Use of Laboratory Animals. Technical specifications can be found in the Table of Materials.
1. Animal handling procedure
<ol>
	<li>House mice in a controlled environment with a temperature of 22-24 &#176;C, humidity ranging from 40%-60%, a 12 h light/dark cycle, and provide ad libitum access to water and standard mouse chow. For the purpose of this experiment, 25 ICR male mice (25-30 g, 8 weeks old) were used.</li>
	<li>Allocate randomly the mice into either the control group (n=12) or the rmTBI group (n=13). To prevent potential aggression from sham mice towards mice those that underwent rmTBI, separate them in distinct cages.</li>
	<li>Give mice at least 1 week to acclimate to their cage environment before beginning the experiment. This acclimation period ensures that the mice become familiar with their surroundings and minimizes the potential impacts of stress or anxiety on physiological or behavioral responses during the study.</li>
</ol>
2. Preparation of TBI device
<ol>
	<li>Fabricate the rmTBI model in mice using a modified FPI device (see Table of Materials; Figure 1A)13. Before using the FPI device, carefully inspect all connections for signs of leakage or cracking, paying special attention to the junctions between the cylinder and tubing, and between the three-way connector and tubing. To determine impact pressures accurately, a&#160;pressure transducer was installed to the point&#160;of impact on the fluid percussion injury device13.</li>
	<li>Conduct a careful inspection of the FPI device to ensure that the piston moves smoothly inside the cylinder and that impacts can be conducted effectively (Figure 1B). Verify that there are no air bubbles present within the system. If air bubbles are detected, carefully add distilled water to the system using a 50 mL syringe and expel the air bubbles by quickly pushing the cylinder rod through a nearby 3-way connector and/or through the syringe. 
	NOTE: The FPI system consists of distilled water within the cylinder and connected tubing. It is crucial to calibrate the device prior to operation to ensure accuracy and reliability of results.</li>
</ol>
3. Thinned-skull preparation
NOTE: The animal surgery and thinned-skull preparation should not be performed in view of other mice. The thinned-skull window is useful for evaluating cerebral vessel damage following an FPI procedure.
<ol>
	<li>Adhere to the requirements of the animal center by instructing the experimental operator to wear a sterile gown and mask during all animal handling procedures.</li>
	<li>Sanitize the lab bench top, FPI device, anesthetic tubing and adjacent counterspace before start experiment using a 70% ethanol spray.</li>
	<li>Weigh the mice in both the sham and rmTBI groups before surgery and compare their weights to those recorded 1 week prior to the experiment. Exclude from the study any mouse displaying poor health such as rough fur coat, diarrhea, weight loss, or lethargy. Additionally, exclude mice that weigh less than 20 g to ensure their ability to tolerate repeated impacts. These measures are crucial in maintaining animal welfare and ensuring reliable experimental outcomes.</li>
	<li>Anesthetize mice with 4%-5% isoflurane (in 100% oxygen at a flow rate of 1 L/min) for 3-4 min in an induction chamber. Administer an ophthalmic lubricant (see Table of Materials) to the animal&#39;s eyes to maintain lubrication throughout the surgery. To mitigate pain, administer buprenorphine subcutaneously (0.05 mg/kg) at the midpoint between their ears 20 min prior to anesthesia and subsequently every 6 h for a duration of 24 h. Furthermore, at the end of anesthesia, administer a single subcutaneous dose of 5 mg/kg carprofen to each animal.</li>
	<li>Disinfect the head after the mouse has lost its righting and pedal withdrawal reflexes. Trim the fur on the mouse&#39;s head using surgical scissors and use a shaver to remove the remaining fur. Disinfect the scalp with three sequential applications of 2% chlorhexidine, interspersed with 75% ethanol scrubs.</li>
	<li>Place a disposable sterile surgical pad under the animal and the surrounding area to ensure proper hygiene. Make a 1.5 cm incision using a fine small surgical scissor along the midline of the mouse&#39;s scalp to fully expose the surgical site (Figure 2A).</li>
	<li>Secure the mouse using nonterminal ear bars in a conventional stereotaxic frame (see Table of Materials). Adjust the mouse head position in a stereotaxic frame at a flat level or a slight tilted angle according to the specific target area that will be investigated. Clean fur from the surgical area to avoid inflammation later.</li>
	<li>Maintain the mouse&#39;s body temperature at 37 &#176;C using a conventional isothermal heating pad (see Table of Materials).</li>
	<li>During the surgical and impact hub (female Luer Lock) installation process, maintain mouse anesthesia (with no response to toe or tail pinch) with a nose cone that delivers a continuous 2% isoflurane concentration, regulated by a calibrated vaporizer.</li>
	<li>Clean the surgical area around the thinned-skull window carefully using a sterile saline-soaked cotton swab.</li>
	<li>Create a thinned-skull window, approximately 2.5 mm in diameter and 20 &#181;m in thickness, in the right frontal motor cortex using a flat-tipped micro-drill bit (see Table of Materials, Figure 2B) and a microsurgical blade. Locate the surgical site at 1.5 mm anterior to the bregma and 1.3-2.0 mm lateral to the midline (Figure 2C).
	<ol>
		<li>To prevent the micro-drill from penetrating the skull during the creation of the thinned-skull window, intermittently moisten the skull with saline while grinding with the micro-drill.</li>
		<li>Confirm the thickness of the skull by gently probing the thinned-skull with the flattened tip of a fine syringe needle and assessing its softness. Estimate the clarity of the exposed cortical micro-vessels visually to determine the thickness of the skull.</li>
		<li>To verify the thickness of the skull, apply sterile saline to the thinned area and visually inspect using a conventional dissecting microscope (see Table of Materials). This technique can help ensure that the skull has been adequately thinned. 
		NOTE: Lubricate the mouse eye throughout the entire thinned-skull preparation and rmTBI modeling procedure to prevent drying out. Thinning the skull to less than 15 &#181;m carries the risk of mild cortical trauma which may result in mild cortical inflammation14.</li>
	</ol>
	</li>
	<li>Attach an adjusted female Luer Lock (2.2 mm inside diameter, created from a 19G needle hub, as shown in Figure 2D) to the thinned-skull site. Secure the Luer Lock with glue and dental cement (Figure 2E). 
	NOTE: When using glue to secure the female Luer lock to the area around the thinned-skull window, it is crucial to thoroughly dry the skull area, and prevent the adhesive from entering the window itself. Adhesive within the window can significantly reduce the impact force of the FPI.</li>
</ol>
4. CHI associated rmTBI modeling procedure
<ol>
	<li>Introduce rmTBI using the lateral fluid percussion method with a modified FPI device as described previously13,15.</li>
	<li>After completing the thinned-skull window and impact hub procedures, transfer the mouse from the stereotaxic apparatus to the impactor platform.</li>
	<li>Given the potential effects of anesthesia on animal righting reflex time and injury severity after percussion9,16, monitor the anesthesia depth by evaluating the palpebral and paw withdrawal reflexes (Figure 2F).</li>
	<li>Connect the female Luer Lock, which was glued onto the thinned-skull window, to the male Luer Lock at the end of the FPI device tubing (Figure 2G). 
	NOTE: In the rmTBI modeling, the isoflurane-induced anesthesia in mice was prolonged due to the induction of apnea and unconsciousness by percussion.</li>
	<li>Introduce two mild TBI (48 h interval) with the modified device. Apply the first FPI impact immediately after completing the thinned-skull window surgery and installing the Luer Lock. Only administer the FPI impact once the mouse shows return of a withdrawal reflex to a paw pinch on each occasion (Figure 2H). Applying an FPI impact in deeply anesthetized mice can cause prolonged apnea and death.
	<ol>
		<li>To apply the FPI impact, raise the pendulum to the designated degree along the protractor on the device and release the pendulum using software control13,15. The impact should achieve a percussion intensity of 2.0 &#177; 0.1 atm, following established protocols used in rodent studies10,17,18. Exclude animals from further tests if the impact did not register between 1.9-2.1 atm or if skull fracture occurred during FPI.</li>
		<li>For the sham mice, fix them onto the apparatus, but do not deliver the impact.</li>
	</ol>
	</li>
	<li>Following impact, immediately detach the Luer Lock connection, and transfer mice to an isothermal heating pad for recovery. After the mouse regains alertness and consciousness, return it to its home cage without removing the female Luer lock. Administer the second FPI impact, in the same manner, 48 h later.</li>
	<li>After the rmTBI, carefully remove the female Luer lock and dental cement. Suture the scalp using tissue adhesive and use flat forceps to pinch the scalp to facilitate the adhesive process (see Table of Materials; Figure 2I).</li>
	<li>To prevent inflammation, infection, and alleviate post-surgical pain and discomfort, apply a mixture of erythromycin and sodium diclofenac ointment in a 1:1 ratio (see Table of Materials) to the wound. Transfer mice to an isothermal heating pad for recovery.</li>
	<li>Record the duration of the righting reflex, which starts when the mouse is removed from the stereotaxic apparatus and placed laterally on the impactor platform for FPI and continues until the mouse can stand upright independently.</li>
	<li>After the mouse regains alertness and consciousness, return it to its home cage. Mice are typically fully conscious and able to walk within 1.5 h after the injury.</li>
	<li>In the days following TBI modeling, observe the mice for various signs including breathing patterns, presence of mucus around the nose and mouth, and redness, swelling, exudates, or reopening of the wound area. Exclude animals from the study with one or more of the above abnormal symptoms. 
	NOTE: Pre-microinjection of AAV-GCaMP6s allows for observation of underlying neuronal Ca2+ homeostasis and excitability in the injured cortex through the thinned-skull window using two-photon laser scanning microscopy15.</li>
</ol>
5. Morris water maze (MWM) test
NOTE: The MWM (see Table of Materials) is a widely recognized method for evaluating spatial learning and memory deficits in mice following TBI.
<ol>
	<li>Conduct the MWM test starting from 7 days post injury (DPI). The circular pool of the MWM had a diameter of 120 cm and a height of 50 cm, with the water temperature maintained at 25 &#176;C. Separate the circular pool into four quadrants, with the escape platform, a round platform with a diameter of 6 cm and a height of 30 cm, submerged 1 cm beneath the water surface in the northeast quadrant.</li>
	<li>Position a camera directly above the circular pool to record the movement trajectory of the mice. Mark the mice with black tape on their backs to facilitate recognition by the image acquisition software and for data recording, including the latency, swimming distance and the movement trajectory.</li>
	<li>Place the mice into the water, facing the interior wall of each of the four quadrants, one time for each quadrant. Once the mice find the platform, allow them to rest there for 10 s. If a mouse fails to find the platform within 60 s, ask the operator to guide the mouse to the platform, allow it to rest on the platform for 10 s, and then return the mouse to its home cage for rest.</li>
	<li>Repeat for each mouse the acquisition trial 4x daily. Following the acquisition trials, on 12 DPI, conduct a 60 s spatial probe experiment and record the number of times that the mice crossed the original platform area and the duration of the mouse stay in the quadrant where the platform was located.</li>
	<li>After each trial, quickly dry the mice with a towel or place under a warming lamp to maintain their body temperature and prevent hypothermia during the 60 s acquisition trial from DPI 7 to DPI 11.</li>
	<li>After the completion of the experimental procedures outlined above, anesthetize the mice with pentobarbital (45 mg/kg, i.p.) at 13 DPI. Perfuse isotonic saline transcardially, followed by perfusion with 4% paraformaldehyde in phosphate-buffered saline (pH 7.2). Retrieve the brains for conventional HE staining to evaluate gross cortical and hippocampal morphology alterations. A detailed description of the HE staining protocol can be found in prior publications13,15.</li>
	<li>After all experiments have been completed, euthanize mouse by injection of overdose pentobarbital (&#8805;100 mg/kg, i.p.) if there are no mouse samples needed. Before harvesting tissues or disposing of the carcass, monitor the mouse until there is no heartbeat for at least 60 s.</li>
</ol>

rmTBI Induction in Mice via a Closed-Head Injury Method

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The authors have no conflicts of interest to disclose.

TBI refers to two primary types, closed and penetrating, with the latter characterized by a disruption of the skull and dura mater. Clinical data suggest that CHIs are more prevalent than penetrating injuries1,2. After a single mild TBI, most patients experience PCS symptoms that typically resolve in a short period of time, and there is controversy regarding the proportion of patients whose PCS develop into long-term sequelae23,...

Mild TBI, including concussion and sub-concussion, account for the majority of all TBI cases (&#62;80% of all TBI)1. Mild TBI commonly results from falls, traffic accidents, acts of violence, contact sports (e.g., football, boxing, hockey), and military combat2,3. Mild TBI can lead to neurobiological events that affect neurobehavioral functions throughout the patient&#39;s lifetime and increase the risk of neurodegenerative diseases4,5,6. Animal models provide an efficient and controlled means to study mild TBI, with the hope of further enhancing the diagnosis and treatment of mild TBI. Various models for mild TBI have been developed, such as the controlled cortical impact (CCI), weight drop (WD), fluid percussion injury (FPI), and blast-TBI models7,8. No single experimental model can mimic the entire complexity of TBI-induced pathology9,10. The heterogeneity of these models is advantageous for addressing the diverse features associated with mild TBI patients and investigating the corresponding cellular and molecular mechanisms. However, each animal model of TBI has its limitations3, limiting our current knowledge concerning animal mild TBI and their clinical relevance.
The WD and CCI models are utilized to replicate clinical conditions such as cerebral tissue loss, acute subdural hematoma, axonal injury, brain concussion, blood-brain barrier dysfunction, and even coma following TBI3,11,12. The WD model involves inducing brain damage by striking either the dura mater or skull with freely falling weights. The impact of a weighted object upon an intact skull can replicate mixed focal/diffuse injuries; however, this method is associated with poor accuracy and repeatability of the injury site, rebound injury, and a higher mortality rate due to skull fractures3,11,12. The CCI model involves applying air-driven metal to impact the exposed dura mater directly. Compared to the WD model, the CCI model is more accurate and reproducible, but it does not produce diffuse injury due to the small diameter of the impacting tip11. During FPI modelling, the brain tissue is briefly displaced and deformed by percussion. FPI can induce mixed focal/diffuse injury and replicate intracranial hemorrhage, brain swelling and progressive gray matter damage after TBI. However, FPI has a high mortality rate due to brainstem damage and prolonged apnea3,12. The craniotomy involved in conventional WD, CCI, and FPI models can lead to cortical contusion, hemorrhagic lesions, the damage of the blood-brain barrier, immune cell infiltration, glial cell activation, prolonged modeling time, and possible fatal outcomes3,12.
Mild TBI is characterized by a GCS (Glasgow coma scale, GCS) score within the range of 13 to 152. Mild TBI can be either focal or diffuse and is associated with both acute injuries, such as breakdown of cellular homeostasis, excitotoxicity, glucose depletion, mitochondrial dysfunction, blood flow disturbance, and axonal damage, as well as subacute injuries, including axonal damage, neuroinflammation, and gliosis2,3. Despite significant progress in delineating the intricate pathophysiology of TBI, the underlying mechanisms of mild TBI/rmTBI remain elusive and require further investigation9. Given that CHI is the most common type of TBI12, this protocol presents a novel approach to creating a more precisely controlled mouse model of rmTBI using a modified FPI device to perform impact in a thinned-skull window13. By avoiding craniotomy-induced injuries, variable skull thickness and shape-induced inaccuracies, and rebound injury, this approach aims to overcome the main disadvantages associated with the WD, CCI, and FPI models. Applying FPI impact on the thinned-skull window is convenient for evaluating cerebral vessel damage following rmTBI and helps minimize high mortality rates in some models, resulting in a closer resemblance to the clinical features of TBI patients.

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			<td>Jiaxing Bocom Biotech Inc.</td>
			<td>Biocom Animal Brain Impactor V1.0</td>
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			<td>Jinhua Laboratory Animal Center&#160;&#160;</td>
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			<td>Shandong Ante Animal Husbandry Technology Co., Ltd.&#160;</td>
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			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Modified FPI device</td>
			<td>Jiaxing Bocom Biotech Inc.</td>
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			<td>Morris water maze</td>
			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Evaluate mouse spatial learning and memory abilities</td>
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			<td>Open field</td>
			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Evaluate mouse locomoation and anxiety</td>
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			<td>Shenzhen RWD Life Science Inc.</td>
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			<td>Smart video-tracking system</td>
			<td>Panlab Harvard Apparatus Inc., MA, USA</td>
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			<td>Stereotactic frame&#160;</td>
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modified mouse model of repetitive mild traumatic brain injury incorporating thinned-skull window and fluid percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models with well-defined pathologies are urgently needed for studying the mechanisms of neuropathology after mild TBI and testing therapeutics. Replicating the entire sequelae of TBI in animal models has proven to be a challenge. Therefore, the availability of multiple animal models of TBI is necessary to account for the diverse aspects and severities seen in TBI patients. CHI is one of the most common methods for fabricating rodent models of rmTBI. However, this method is susceptible to many factors, including the impact method used, the thickness and shape of the skull bone, animal apnea, and the type of head support and immobilization utilized. The aim of this protocol is to demonstrate a combination of the thinned-skull window and fluid percussion injury (FPI) methods to produce a precise mouse model of CHI-associated rmTBI. The primary objective of this protocol is to minimize factors that could impact the accuracy and consistency of CHI and FPI modeling, including skull bone thickness, shape, and head support. By utilizing a thinned-skull window method, potential inflammation due to craniotomy and FPI is minimized, resulting in an improved mouse model that replicates the clinical features observed in patients with mild TBI. Results from behavior and histological analysis using hematoxylin and eosin (HE) staining suggest that rmTBI can lead to a cumulative injury that produces changes in both behavior and gross morphology of the brain. Overall, the modified CHI-associated rmTBI presents a useful tool for researchers to explore the underlying mechanisms that contribute to focal and diffuse pathophysiological changes in rmTBI.

The protocol described in this study outlines a method for inducing rmTBI through a thinned-skull window, which offers a solution to the brain injury caused by craniotomy preparation during conventional percussion TBI modeling. By utilizing this modified fluid percussion procedure with the modified device, improved precision and reproducibility of FPI impact were achieved13. The modified impactor has the versatility to be used for both CHI and FPI modeling, with or without a skull craniotomy. Furt...

Read this Scientific Article Protocol about Author Spotlight: Optimizing Rodent Models for Investigating Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury at JoVE.com

Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Modified Mouse Model of Repetitive Mild Traumatic Brain Injury Incorporating Thinned-Skull Window and Fluid Percussion

Mild traumatic brain injury is a clinically highly heterogeneous neurological disorder. Highly reproducible traumatic brain injury (TBI) animal models ...

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302 Views.  Zhejiang Normal University. This protocol presents a modified mouse model of repetitive mild traumatic brain injury (rmTBI) induced via a closed-head injury (CHI) method. The approach features a thinned-skull window and fluid percussion to reduce the inflammation commonly caused by meninges exposure, along with improved reproducibility and accuracy in modeling rmTBI in rodents.

Video: Author Spotlight: Optimizing Rodent Models for Investigating the Mechanisms and Rehabilitation in Repeated Mild Traumatic Brain Injury

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

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

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

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

A Polished and Reinforced Thinned-skull Window for Long-term Imaging of the Mouse Brain

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 &mu;m in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 &mu;m below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

We present a method to form an imaging window in the mouse skull that spans millimeters and is stable for months without inflammation of the brain. This method is well suited for longitudinal studies of blood flow, cellular dynamics, and cell/vascular structure using two-photon microscopy.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form ...

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

Optical coherence tomography (OCT) is a biomedical imaging technique with high spatial-temporal resolution. With its minimally invasive approach OCT has been used extensively in ophthalmology, dermatology, and gastroenterology1-3. Using a thinned-skull cortical window (TSCW), we employ spectral-domain OCT (SD-OCT) modality as a tool to image the cortex in vivo. Commonly, an opened-skull has been used for neuro-imaging as it provides more versatility, however, a TSCW approach is less invasive and is an effective mean for long term imaging in neuropathology studies. Here, we present a method of creating a TSCW in a mouse model for in vivo OCT imaging of the cerebral cortex.

Thinned-skull Cortical Window Technique for In Vivo Optical Coherence Tomography Imaging

We present a method of creating a thinned-skull cortical window (TSCW) in a mouse model for in vivo OCT imaging of the cerebral cortex.

Optical coherence tomography (OCT) is a biomedical imaging technique  with high spatial-temporal resolution. With its minimally invasive approach OCT  has ...

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 ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

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

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

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

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

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 ...