The authors would like to thank Custom Design &#38; Fabrication Inc. for their technical assistance and support. This work was funded by the National Institutes of Health grants R01NS120099-01A1 and R37HD059288-19.

1. Cleaning the LFPI cylinder
<ol>
	<li>Carefully detach the syringes attached to the transducer housing and fill port, as well as the cable connected to the pressure transducer (see Figure 1 for a schematic of injury device components).</li>
	<li>While being careful not to drop the cylinder, unscrew the hand knobs at the back of the device from the cylinder clamps to free the cylinder.</li>
	<li>Remove the piston at the end of the cylinder, transducer, transducer housing, and plunger O-rings.</li>
	<li>Drain fluid out of the cylinder.</li>
	<li>Add mild detergent, such as dish washing detergent, to the cylinder and scrub lightly using a dish or bottle brush13.</li>
	<li>To ensure that all the detergent is rinsed off, completely fill the cylinder with water and rinse thoroughly.</li>
</ol>
2. Degassing the fluid used to fill the cylinder
<ol>
	<li>Use a vacuum pump to degas the fluid before refilling the cylinder to prevent the formation of new bubbles and absorb existing bubbles. 
	NOTE: Approximately 1.5 L of fluid will be needed to fill the cylinder, although degassing approximately 2 L will leave a small supply for replacing any fluid lost during use and testing. 
	NOTE: House vacuums are too weak to effectively degas the fluid. The vacuum must be able to produce a pressure of 25-28 inHg.</li>
	<li>Add a stir bar to the fluid and place the fluid container on a stir plate. Stirring the fluid during the degassing process helps to stimulate bubbling and the release of gas. Stirring also prevents a large sudden increase in bubbling. 
	&#8203;NOTE: The degassing process should be over when very few bubbles are being produced; this occurs after roughly 45 min.</li>
</ol>
3. Reassembly of the LFPI device
<ol>
	<li>Apply a thin layer of petroleum jelly to the piston plunger.</li>
	<li>Attach the piston plunger with the plunger protruding approximately 32 mm from the cylinder14. 
	NOTE: Air frequently gets trapped at the plunger before the leading O-ring. To get rid of this excess air, twist the plunger while moving it in and out to work the air out of this gap.</li>
	<li>Apply a thin layer of petroleum jelly to the other O-rings as well and attach them to the cylinder, with exception to the O-ring on the fill port.</li>
	<li>Wrap Teflon tape twice around the transducer&#39;s threads.</li>
</ol>
4. Refilling the LFPI device and attachment to the base
<ol>
	<li>Connect a 10 mL syringe filled with degassed fluid and free of air bubbles to the Luer lock hub on the transducer housing.</li>
	<li>Hold the transducer with the threaded end pointing upward, and completely fill the well inside the threaded region of the transducer with the degassed fluid using a 10 mL syringe. The goal here is to fill the transducer well without introducing any air bubbles. Be careful not to damage the delicate membrane at the bottom of the transducer well.</li>
	<li>With the cylinder placed at an angle to prevent air from reentering the transducer housing, attach the transducer housing to the cylinder13 and use a wrench to tighten it snugly.</li>
	<li>Remove the cap from the fill port and cylinder once the degassed fluid reaches approximately 2/3 of the cylinder capacity.</li>
	<li>Place the cylinder horizontally and finish filling the cylinder with degassed fluid. 
	NOTE: To avoid the formation of air bubbles, pouring the fluid in slowly is recommended14.</li>
	<li>Replace the cap at the fill port and close all stopcocks.</li>
	<li>Manipulate the cylinder to work any air bubbles to the fill port14.</li>
	<li>Open the stopcock on the fill port and inject fluid using the syringe on the transducer housing to force any air bubbles out of the port14.</li>
	<li>Inspect the entire device and ensure that there are no air bubbles.</li>
	<li>Add a 10 mL syringe filled with degassed fluid to the Luer lock hub on the fill cap.</li>
	<li>Reattach the cylinder to the base using the hand screws.</li>
	<li>Ensure that the cylinder is horizontal and lined up with the center of the weighted hammer on the pendulum.</li>
</ol>

<ol>
	<li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+Report+of+Traumatic+Brain+Injury-related+Emergency+Department+Visits,+Hospitalizations,+and+Deaths.">Surveillance Report of Traumatic Brain Injury-related Emergency Department Visits, Hospitalizations, and Deaths.</a> Centers for Disease Control and Prevention, U.S. Department of Health and Human Services. , (2014).</li><li>Dewan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+global+incidence+of+traumatic+brain+injury.">Estimating the global incidence of traumatic brain injury.</a> Journal of Neurosurgery. 130 (4), 1080-1097 (2018).</li><li>National Center for Injury Prevention and Control; Division of Unintentional Injury Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Traumatic Brain Injury in the United States: Epidemiology and Rehabilitation. , (2015).</li><li>Holm, L., Cassidy, J. D., Carroll, L. J., Borg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Summary+of+the+WHO+Collaborating+Centre+for+neurotrauma+task+force+on+mild+traumatic+brain+injury.">Summary of the WHO Collaborating Centre for neurotrauma task force on mild traumatic brain injury.</a> Journal of Rehabilitation Medicine. 37 (3), 137-141 (2005).</li><li>Pavlovic, D., Pekic, S., Stojanovic, M., Popovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury:+neuropathological,+neurocognitive+and+neurobehavioral+sequelae.">Traumatic brain injury: neuropathological, neurocognitive and neurobehavioral sequelae.</a> Pituitary. 22 (3), 270-282 (2019).</li><li>Dixon, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluid+percussion+model+of+experimental+brain+injury+in+the+rat.">A fluid percussion model of experimental brain injury in the rat.</a> Journal of Neurosurgery. 67 (1), 110-119 (1987).</li><li>McIntosh, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+brain+injury+in+the+rat:+characterization+of+a+lateral+fluid-percussion+model.">Traumatic brain injury in the rat: characterization of a lateral fluid-percussion model.</a> Neuroscience. 28 (1), 233-244 (1989).</li><li>Ma, X., Aravind, A., Pfister, B. J., Chandra, N., Haorah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injury+and+assessment+of+injury+severity.">Animal models of traumatic brain injury and assessment of injury severity.</a> Molecular Neurobiology. 56 (8), 5332-5345 (2019).</li><li>Nwafor, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+traumatic+brain+injury:+an+update+on+preclinical+models,+clinical+biomarkers,+and+the+implications+of+cerebrovascular+dysfunction.">Pediatric traumatic brain injury: an update on preclinical models, clinical biomarkers, and the implications of cerebrovascular dysfunction.</a> Journal of Central Nervous System Disease. 14, (2022).</li><li>Turner, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+chronic+traumatic+encephalopathy:+the+way+forward+for+future+discovery.">Modeling chronic traumatic encephalopathy: the way forward for future discovery.</a> Frontiers in Neurology. 6, 223 (2015).</li><li>Petersen, A., Soderstrom, M., Saha, B., Sharma, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+traumatic+brain+injury:+a+review+of+pathophysiology+to+biomarkers+and+treatments.">Animal models of traumatic brain injury: a review of pathophysiology to biomarkers and treatments.</a> Experimental Brain Research. 239 (10), 2939-2950 (2021).</li><li>Sullivan, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid-percussion+model+of+mechanical+brain+injury+in+the+cat.">Fluid-percussion model of mechanical brain injury in the cat.</a> Journal of Neurosurgery. 45 (5), 521-534 (1976).</li><li>Pernici, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+optical+imaging+technique+to+visualize+progressive+axonal+damage+after+brain+injury+in+mice+reveals+responses+to+different+minocycline+treatments.">Longitudinal optical imaging technique to visualize progressive axonal damage after brain injury in mice reveals responses to different minocycline treatments.</a> Scientific Reports. 10, 7815-78 (2020).</li></ol>

No conflicts of interest declared.

The techniques outlined above demonstrate how to properly maintain an LFPI device. Routine cleaning and monitoring are necessary to keep the LFPI device functioning correctly and reliably. Additionally, due to the invasive nature of the LFPI procedure, it is imperative that the device be cleaned thoroughly to prevent infection of laboratory animals. 
Avoiding the formation of air bubbles in the device is crucial for obtaining optimal injuries and pressure waveforms. Air bubbles alter the chara...

Traumatic brain injury (TBI) is caused by a sudden force applied to the head. Following primary injuries resulting from the physical impact, TBI survivors commonly experience secondary injuries, including cognitive deficits and neurological dysfunctions that are associated with physiological responses to the initial injury1. It is estimated that roughly 69 million individuals worldwide suffer from TBI annually2. In the United States alone, approximately 2.5 million TBI-related emergency room visits and hospitalizations occur each year, making TBI one of the leading causes of disability and death among children and young adults3. TBI can be classified as mild, moderate, or severe, with mild TBI (mTBI) accounting for approximately 70%-90% of TBI cases4. Histological and cognitive TBI pathology can occur within minutes to hours of injury, and the effects of TBI can persist for months to years after initial damage5.
The development of experimental models has been instrumental in understanding the effects and underlying mechanisms of TBI. One such model, the lateral fluid percussion injury (LFPI), is commonly used to assess TBI in vivo. LFPI closely reproduces pathologies associated with human TBI, including vascular disruptions, hemorrhages, neuronal loss, inflammation, gliosis, and molecular disturbances6,7,8. The LFPI technique is used for a diverse set of experimental applications, including modeling pediatric TBI, as well as chronic neurodegenerative conditions, such as chronic traumatic encephalopathy9,10. LFPI is a well-defined and reproducible method of experimental TBI that allows for the severity of the injury to be adjusted11. The LFPI device has several important components, including: a pendulum with a weighted hammer, a piston, a fluid-filled cylinder, a pressure transducer, a digital oscilloscope, and a small tube at the end of the cylinder with a Luer lock which attaches to a hub on the animal&#39;s skull (Figure 1). LFPI works by swinging the pendulum into the piston, creating a wave of pressure through the fluid (degassed deionized water or saline) into the brain of the attached animal; this increases intracranial pressure, thus replicating the mechanical features and biological changes of TBI12. Additionally, animals used in LFPI experiments undergo a craniectomy in order to expose the brain to the impact of the fluid pressure of the device.
Routine maintenance and monitoring are necessary to ensure that the LFPI device is accurately functioning. The following methods are vital in preventing the introduction of contaminating air bubbles into the device. Here, we demonstrate methods to properly clean, fill, and assemble the LFPI device. We will also discuss oscilloscope outputs and mouse righting times as ways to confirm the viability of the LFPI.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2 - 10 mL syringes with Luer lock capability</td>
			<td></td>
			<td></td>
			<td>Ensures that needle is secure and reduces possible leaks of fluid&#160;</td>
		</tr>
		<tr>
			<td>Degassed fluid</td>
			<td></td>
			<td></td>
			<td>Helps to reduce air bubble formation during injury procedure</td>
		</tr>
		<tr>
			<td>Fluid Percussion Injury (FPI) device (Model 01-B)</td>
			<td>Custom Designs &#38; Fabrications Inc.</td>
			<td>N/A</td>
			<td>Injury device used to model TBI in rodents</td>
		</tr>
		<tr>
			<td>Mild detergent</td>
			<td></td>
			<td></td>
			<td>Allows to thoroughly clean the LFPI cylinder&#160;</td>
		</tr>
		<tr>
			<td>Petroleum Jelly</td>
			<td></td>
			<td></td>
			<td>Used as a water-repellent and protects LFPI device form rust</td>
		</tr>
		<tr>
			<td>Teflon tape</td>
			<td></td>
			<td></td>
			<td>Helps with tight seal of pipe joints on the LFPI device</td>
		</tr>
		<tr>
			<td>*Materials other than the LFPI device can be purchased from any reliable company.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

maintenance of a lateral fluid percussion injury device

Traumatic brain injury (TBI) accounts for roughly 2.5 million emergency room visits and hospitalizations annually and is a leading cause of death and disability in children and young adults. TBI is caused by a sudden force applied to the head and, to better understand human TBI and its underlying mechanisms, experimental injury models are necessary. Lateral fluid percussion injury (LFPI) is a commonly used injury model due to similarities in the pathological changes found in human TBI compared to LFPI, including hemorrhages, vascular disruption, neurological deficits, and neuron loss. LFPI employs a pendulum and a fluid-filled cylinder, the latter having a moveable piston at one end, and a Luer lock connection to stiff, fluid-filled tubing at the other end. Preparation of the animal involves performing a craniectomy and attaching a Luer hub over the site. The next day, the tubing from the injury device is connected to the Luer hub on the animal's skull and the pendulum is raised to a specified height and released. The impact of the pendulum with the piston generates a pressure pulse which is transmitted to the intact dura mater of the animal via the tubing and produces the experimental TBI. Proper care and maintenance are essential for the LFPI device to function reliably, as the character and severity of the injury can vary greatly depending on the condition of the device. Here, we demonstrate how to properly clean, fill, and assemble the LFPI device, and ensure that it is adequately maintained for optimal results.

We tested the effects of air bubble contamination in an LFPI device on waveform formation. We injected air bubbles into the device and compared the oscilloscope outputs with oscilloscope data collected from a non-contaminated LFPI device. Conditions were as follows: non-contaminated, injection of 5 mL of air, injection of 10 mL of air, and injection of 15 mL of air. We kept the pendulum at a consistent height for all impacts for all conditions, and we performed 15 impacts per condition.
When p...

Watch this Scientific Journal Video about Maintenance of a Lateral Fluid Percussion Injury Device at JoVE.com

Maintenance of a Lateral Fluid Percussion Injury Device

Proper care and maintenance are essential for a lateral fluid percussion injury (LFPI) device to function reliably. Here, we demonstrate how to properly clean, fill, and assemble an LFPI device, and ensure that it is adequately maintained for optimal results.

Traumatic brain injury (TBI) accounts for roughly 2.5 million emergency room visits and hospitalizations annually and is a leading cause of death and ...

maintenance-of-a-lateral-fluid-percussion-injury-device

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800 Views.  Children’s Hospital of Philadelphia. Proper care and maintenance are essential for a lateral fluid percussion injury (LFPI) device to function reliably. Here, we demonstrate how to properly clean, fill, and assemble an LFPI device, and ensure that it is adequately maintained for optimal results. 

Video: Maintenance of a Lateral Fluid Percussion Injury Device

Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been generally adopted to study the nature of neural stem cells, including those derived from normal and tumorous tissues. However, appreciable amounts of differentiation and cell death are common in cultured neurospheres likely due to sub-optimal condition such as accessibility of all cells within sphere aggregates to culture medium.
Medulloblastoma, the most common pediatric CNS tumor, is characterized by its rapid progression and tendency to spread along the entire brain-spinal axis with dismal clinical outcome. Medulloblastoma is a neuroepithelial tumor of the cerebellum, accounting for 20% and 40% of intracranial and posterior fossa tumor in childhood, respectively1. It is now well established that Shh signaling stimulates proliferation of cerebellar granule neuron precursors (CGNPs) during cerebellar development 2-4. Numerous studies using mouse models, in which the Shh pathway is constitutively activated, have linked Shh signaling with medulloblastoma 5-9.
A recent report has shown that a subset of medulloblastoma cells derived from Patched1LacZ/+ mice are cancer stem cells, which are capable of initiating and propogating tumors 10. Here we describe an efficient method to isolate, enrich and maintain tumor stem cells derived from several mouse models of medulloblastoma, with constitutively activated Shh pathway due to a mutation in Smoothened (11, hereon referred as SmoM2), a GPCR that is critical for Shh pathway activation. In every isolated medulloblastoma tissue, we were able to establish numerous highly proliferative colonies. These cells robustly expressed several neural stem cell markers such as Nestin and Sox2, can undergo serial passages (greater than 20) and were clonogenic. While these cultured tumor stem cells were relatively small, often bipoar with high nuclear to cytoplasmic ratio when cultured under conditions favoring stem cell growth, they dramatically altered their morphology, extended multiple cellular processes, flattened and withdrew from the cell cycle upon switching to a cell culture medium supplemented with 10% fetal bovine serum. More importantly, these tumor stem cells differentiated into Tuj1+ or NeuN+ neurons, GFAP+ astrocytes and CNPase+ oligodendrocytes, thus highlighting their multi-potency. Furthermore, these cells were capable of propagating secondary medulloblastomas when orthotopically transplanted into host mice.

This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.

Brain tumors have been suggested to possess a small population of stem cells that are the root cause of tumorigenesis. Neurosphere assays have been ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

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 ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. 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 ...

Maintenance of a Lateral Fluid Percussion Injury Device

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A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Generation and Long-term Maintenance of Nerve-free Hydra

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