The authors thank Voltaire Gungab for assistance with manuscript preparation. This work was supported in part by grants from: the National Institutes of Health (T32 GM008440 to E.J. Walker, R01 NS27713 to W.L. Young, P01 NS44155 to W.L. Young and H. Su), and R21 NS070153 to H. Su; and the American Heart Association (AHA 10GRNT3130004 to H. Su).

1. Clearing Blood from Vasculature
<ol>
	<li>Anesthetize mouse by intraperitoneal injection of 100 mg/kg ketamine with 10 mg/kg xylazine diluted in 0.25 ml saline.</li>
	<li>Once anesthesia has been confirmed by no response to a paw pinch, use scissors to make an incision across the chest with scissors just below the xyphoid process, cut through diaphragm, and cut up between dorsal and ventral segment of ribcage to expose thoracic cavity. Expose heart by folding up sternum and adjacent chest wall and secure with hemostat. Open the pericardial sac with tweezers to expose the heart.</li>
	<li>Turn on perfusion pump (100mmHg pressure) connected to warm PBS plus heparin (5 unit/ml) solution in hot water bath at 37&deg;C and tubing with a blunt 22 gauge needle on the outflow.</li>
	<li>Insert the needle into the left ventricle, near the apex of the heart. Immediately cut open the right atrium to enable systemic blood outflow and continue flushing until blood is removed from circulation.</li>
</ol>
2. Perfusion of Casting Agent into the Vascular System
<ol>
	<li>Prepare Microfil (Flow Tech, Inc. Carver, MA) casting solution per manufacturer's protocol: Mix 5 ml of MV diluent with 4 ml of filtered MV compound. Add 450&mu;l (5%) of catalyst (MV curing agent). Solution working time is 20 minutes, but mix immediately before use to ensure proper viscosity.</li>
	<li>Withdraw casting agent solution into a 10ml syringe, attach a blunt 20 gauge needle to the syringe and fill needle with solution.</li>
	<li>Insert needle into the left ventricle through the same hole used for flushing the blood. Secure needle into place with hemostat clamp on the heart.</li>
	<li>Inject casting agent solution slowly into the left ventricle at approximately 3 ml/min. Excess pressure may lead to incorrect flow of casting agent or potential vessel rupture.</li>
	<li>Look for signs of successful perfusion, including: visualization of casting agent in intestine and liver vasculature, distal limb discoloration, and nose and tongue discoloration. Readjust needle if necessary to achieve proper perfusion.</li>
</ol>
3. Collection and Processing of Tissue
<ol>
	<li>Remove brain and place in 4% paraformaldehyde overnight at 4&deg;C.</li>
	<li>Dehydrate brain by placing from paraformaldehyde directly into increasingly concentrated EtOH at room temperature: 25% EtOH, 1 day; 50% EtOH, 1 day; 75% EtOH, 1 day; 95% EtOH, 2 days; and 100% EtOH, 2 days.</li>
	<li>Clarify brain tissue around blood vessels by immersing the perfused brain in methyl salicylate for 2 days at room temperature.</li>
	<li>Vasculature of the whole unsectioned clarified brain can be imaged with brain immersed in methyl salicylate under a dissecting microscope (Fig. 1) (Leica MZFL III microscope, Leica Microsystems, Bannockburn, IL) or brightfield microscope (Fig. 2) (Leica DM/LS, Leica Microsystems).</li>
	<li>Reclarification can be done if tissue is not completely cleared. From methyl salicylate, transfer brain to 95% EtOH for 2 days, 100% EtOH for 2 days, and methyl salicylate for 2 days.</li>
</ol>
4. Micro-CT Imaging or Histological Application- Tissue Preparation
<ol>
	<li>The brain can be imaged using micro-CT directly after clarification or after re-hydration (Fig. 4).</li>
	<li>To prepare brain tissue for histological analysis, rehydrate the brain through serial diluted EtOH: 100% EtOH, 1 day; 95% EtOH, 1 day; 75% EtOH, 1 day; 50% EtOH, 2 days; 25% EtOH, 2 days; and store in PBS. The brain can then be embedded in paraffin using a standard procedure.</li>
</ol>
5. Representative Results
Following vascular casting, the cerebrovasculature can be seen macroscopically on the brain surface (Fig.1A). The vascular tree inside the parenchyma can be visualized following clarification (Fig. 1B). The detailed vascular structure can be imaged and analyzed under a brightfield microscope (Fig. 2), dissecting scope (Fig. 3), and with micro-CT scanning (Fig. 4). Using brightfield microscopy, the vasculature can be viewed at different focal planes without sectioning the brain (Fig. 2A) as well as individual microvessels at higher magnification (Fig. 2B). Under a dissecting scope, one can see the entire vascular structure in one focal plane, allowing better visualization of the three dimensional structure of the cerebrovasculature (Fig. 3). Images taken under a dissecting microscope can show large vessel morphology and whole brain vasculature, but lack the details shown in pictures taken with brightfield microscopy (Fig. 2B). This technique has allowed us to detect irregular vessels in our experimental BAVM adult mouse model2. Figure 5 shows an example of the same irregular vessels detected by micro-CT scanning (Fig. 5A) and brightfield microscope (Fig. 5B). Together, this data shows that we can analyze the same sample with multiple imaging tools after vascular casting.
<img alt="Figure 1" src="/files/ftp_upload/2958/2958fig1.jpg" /> 
Figure 1. Whole mouse brain following vascular casting. Before (A) and after (B) clarification by methyl salicylate solvent. Vessels can be clearly visualized on the brain surface (A) and in parenchyma following clarification procedure (B).
<img alt="Figure 2" src="/files/ftp_upload/2958/2958fig2.jpg" /> 
Figure 2. Brightfield microscope images of clarified vascular casted brain. Brain can be imaged at various planes and at different magnification levels. Scale Bar 200&mu;m (A) and 50&mu;m (B).
<img alt="Figure 3" src="/files/ftp_upload/2958/2958fig3.jpg" /> 
Figure 3. Dissecting microscope image at 1x showing whole adult mouse brain cerebrovasculature from a dorsal view.
<img alt="Figure 4" src="/files/ftp_upload/2958/2958fig4.jpg" /> 
Figure 4. Micro-CT scanning image of the same whole adult mouse brain shown in Figure 3 from a dorsal view.
<img alt="Figure 5" src="/files/ftp_upload/2958/2958fig5.jpg" /> 
Figure 5. Application of vascular casting method to detect a brain arteriovenous malformation. A) Micro-CT scan of vascular casted brain shows region of enlarged, irregular AVM-like vessels in right hemisphere from a ventral view. Enlarged image of region (white box) is shown on the right. B) Same irregular vessels visualized under brightfield microscope at 50x magnification. Scale bar 50 &mu;m.

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	<li>Bolland, B. J., Kanczler, J. M., Dunlop, D. G., Oreffo, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+in+vivo+muCT+evaluation+of+neovascularisation+in+tissue+engineered+bone+constructs.">Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs.</a> Bone. 43, 195-202 (2008).</li><li>Walker, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriovenous+malformation+in+the+adult+mouse+brain+resembling+the+human+disease.">Arteriovenous malformation in the adult mouse brain resembling the human disease.</a> Ann. Neurol. , (2011).</li><li>Iqbal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+novel+mono-+and+multivalent+VHH-fragments+and+their+application+for+molecular+imaging+of+brain+tumours.">Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.</a> Br. J. Pharmacol. 160, 1016-1028 (2010).</li><li>Fukunaga, A., Kawase, T., Uchida, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+after+simultaneous+transplantation+with+neuro-epithelial+stem+cells+and+adjacent+mesenchymal+tissues+into+infarcted+rat.">Functional recovery after simultaneous transplantation with neuro-epithelial stem cells and adjacent mesenchymal tissues into infarcted rat.</a> 145, 473-480 (2003).</li><li>Chugh, B. 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Experimental procedures for using laboratory animals were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco (UCSF).

We report here a method for casting of adult mouse cerebrovasculature that may be imaged with various modalities. Application of this method to the disease model BAVM enables 3D analysis of irregular vessels. Potential future studies include quantification of micro-CT images, histological analysis of tissue, and diagnosis of vascular disease progression.
Common Problems Encountered and Suggestions
Complete perfusion of the brain vasculature is not always achieved. Having enough pressure to reach the distal blood vessels without rupturing the left atrium or aorta is a challenge. To overcome this, apply consistent pressure of approximately 100mmHg. Also, adjust needle position to ensure appropriate outflow of casting agent into the aorta. It is important that the carotid arteries are not restricted so the casting agent can reach the brain vessels. Critical procedures that correlate with improved small vessel perfusion are: (1) proper clearing of the blood from the vasculature; (2) filtration of the casting agent; and (3) needle placement during casting agent injection. Care should be taken to avoid injection of casting agent into the left atrium and lung. Other investigators have successfully perfused mouse3 and rat4 cerebrovasculature without using additional procedures. Manual analysis of Microfil perfused vessels counterstained with hemotoxylin showed 93% vessel lumen perfusion5. Always discard or completely clean any tool that comes in contact with the casting agent as it will polymerize quickly, and tools cannot be used for multiple subjects. Clarification of the surrounding mouse brain tissue can be challenging. Following immersion in methyl salicylate, the tissue may still appear opaque, making vessel imaging difficult. Ensuring proper dehydration and clarification should solve this problem. Placing brain tissue on a rocker during alcohol and methyl salicylate treatment can improve tissue clarification. 
Drawbacks of the Technique
The limitations of this technique include: (1) it is necessary to inject the casting agent solution immediately after mixing it as it will thicken quickly after the catalyst is added; (2) the casting agent fills large conductance vessels easily, but it does not reliably fill smaller vessels, such as arterioles (resistance vessels) and capillaries (nutritive vessels); and (3) it is not the best radiopaque contrast for micro-CT imaging, although some groups prefer Microfil for micro-CT imaging1,6. However, following the suggestions proposed above, including proper needle placement, vessel position and perfusion pressure, complete casting of smaller vessels can be achieved. The adaptability to various analysis tools makes this agent a valuable tool for analyzing cerebrovascular structure, including arterial and venous circulation, with various perspectives.
Applications of the Technique
The benefit of this method is its broad application potential. The cast fills all vessels, including arteries, veins, and capillaries of all tissue within the body, so vascular structures in multiple organs of various species can be analyzed. Previously, other groups have used this perfusion for large7 and small8 vessels in human, monkey9, sheep10, rat4,11,12, and mouse3,13. We now show evidence of using this technique in analyzing the structure of microvessels in the mouse brain, in both healthy and diseased states2. In addition, there are Microfil solutions with various viscosities available to perfuse vessels of different sizes and different colors to improve the visualization of the vessels in various organs. For example, red can be easily visualized in the pale brain parenchyma, and yellow contrasts well with dark red myocardium. Moreover, as the material is radiopaque, vasculature can be imaged with micro-CT scanning to obtain a 3D rotatable image. The tissue can also be used for paraffin sections and histological analysis after being imaged.
Conclusion
We demonstrate here a protocol to make a vascular cast of the blood vessels of the adult mouse brain, which can be adapted using a variety of analysis techniques to study the morphological structure of the cerebrovasculature. We have also provided evidence for the application of this method to a model of the BAVM disease state, represented by enlarged, irregularly shaped vessels that shunt blood from arterial to venous circulations. There are a variety of vascular casting methods available. The simple protocol we provide here for a vascular cast may be used as a tool to examine the vasculature of the adult mouse brain using various imaging modalities.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>Microfil			</td>
			<td>FlowTech, Inc.</td>
			<td>MV130</td>
			<td>4 month shelf life</td>
		</tr>
		<tr>
			<td>Methyl Salicylate</td>
			<td>Fisher Scientific</td>
			<td>O3695-500</td>
			<td>to clarify tissue</td>
		</tr>
	</tbody>
</table>

cerebrovascular casting of the adult mouse for 3d imaging and morphological analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

Watch this Scientific Journal Video about Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis at JoVE.com

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

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ビデオ: Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

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Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Fluorescence Angiography for Evaluation of Aneurysm Perfusion and Parent Artery Patency in Rat and Rabbit Aneurysm Models

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and indocyanine green are used to enable the observation of blood flow and vessel perfusion status, respectively. The aim of this study is to apply FVA to verify real-time blood flow, vessel perfusion status and occlusion of aneurysms after induction of sidewall aneurysms in rabbits and rats, as well as to validate the procedure in these species.
Twenty sidewall aneurysms were created in 10 rabbits by suturing a decellularized arterial vessel pouch on the carotid artery of a donor rabbit. In addition, 48 microsurgical sidewall aneurysms were created in 48 rats. During follow-up at one month after creation, the parent artery/aneurysm complex was dissected and FVA was performed using an intravenous fluorescein (10%, 1 mL) injection via an ear vein catheterization in rabbits and a femoral vein catherization in rats. Aneurysms were then harvested, and patency was evaluated macroscopically.
Macroscopically, 14 out of 16 aneurysms in rabbits indicated no residual parent artery perfusion with totally occluded luminae, however 11 (79%) were detected by FVA. Four aneurysms were excluded due to technical problems. In rats, residual aneurysm perfusion was macroscopically observed in 25 out of 48 cases. Of the 23 without macroscopic evidence of perfusion, FVA confirmed the incidence of 22 aneurysms (96%). There were no adverse events associated with FVA. Fluorescein is easily applicable and no special equipment is needed. It is a safe and extremely effective method for evaluating parent artery integrity and aneurysm patency/residual perfusion in an experimental setting with rabbits and rats. FVA using fluorescein as a contrast agent appears to be effective in controlling patency of aneurysms and the underlying vessel and can even be adapted to bypass surgery.

We present a protocol to efficiently evaluate aneurysm perfusion and vessel patency of sidewall aneurysm in rats and rabbits, using fluorescein-based fluorescence video angiography (FVA). With a positive predictive value of 92.6%, it is a simple but very effective and economical method with no special equipment required.

Brain aneurysm treatment focuses on achieving complete occlusion, as well as preserving blood flow in the parent artery. Fluorescein sodium and ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...