SL was supported by the Chinese Scholarship Council. KT was supported by the BIH-MD scholarship of the Berlin Institute of Health and the Sonnenfeld-Stiftung. RX is supported by the BIH-Charit&#233; Clinician Scientist Program, funded by the Charit&#233; -Universit&#228;tsmedizin Berlin and the Berlin Institute of Health. We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charite&#769; - Universita&#776;tsmedizin Berlin.

The experiments were performed in accordance with the guidelines and regulations set forth by Landesamt fuer Gesundheit und Soziales (LaGeSo), Berlin, Germany (G0063/18). In this study, C57Bl/6J male (8-12 weeks old) mice with a weight of 25 &#177; 0.286 g (average &#177; s.e.m.) were used.
1. Animal preparation
<ol>
	<li>Induce anesthesia by injecting ketamine (70 mg/kg) and xylazine (16 mg/kg) intraperitoneally. Maintain normal body temperature, contributing to quick induction of deep anesthesia. Test for adequate sedation with a pain stimulus, such as a toe pinch, and verify the absence of a reaction.</li>
	<li>Carefully shave the neck hair of the mouse with a razor, clean it with 70% ethanol followed by betadine/chlorhexidine, and apply 1% lidocaine on the skin surface for local pain control.</li>
	<li>Place the mouse in a supine position. Use tape to fix the limbs and tail, gently stretching the skin of the neck to the opposite side of the surgery. Simultaneously, elevate the neck slightly.</li>
	<li>Use ophthalmic ointment (e.g., 5% dexpanthenol) to prevent dehydration of the eyes during the operation.</li>
</ol>
2. SAH induction
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63150/63150fig01.jpg" /> 
Figure 1: Step-by-step images of surgical technique. (A) Depiction of the exposed right carotid artery anatomy: the CCA and its bifurcation into ICA and ECA are identified, as well as the small branches of the ECA (OA and STA). (B) The ECA is mobilized from the surrounding tissue and ligated with two sutures before cutting it. A third ligation needs to be placed loosely near the bifurcation without occluding it. (C) The ICA and CCA are occluded temporarily (with either ligation or clips) to prevent excessive bleeding when the ECA is carefully incised. (D) The filament is inserted into the ECA and advanced into the CCA. The prearranged ligation must be tightened carefully so that no blood effusion occurs but advancing the filament remains possible. (E) The ICA and CCA are reopened, and the ECA stump needs to be adjusted to a cranial direction. By pushing the filament ~9 mm forward into the ICA, the ACA-MCA bifurcation will be reached, and the vessel is then perforated by pushing the filament ~3mm further. (F) The filament is withdrawn after ensuring a temporal re-ligation of the CCA. The prearranged ligation of the ECA is quickly occluded, and the CCA is reopened to allow reperfusion. Abbreviations: ACA = anterior cerebral artery, CCA = common carotid artery, ECA = external carotid artery, MCA = middle cerebral artery, ICA = internal carotid artery, OA = occipital artery, PPA = pterygopalatine artery, STA = superior thyroid artery. Scale bar = 2 mm. <a href="https://www.jove.com/files/ftp_upload/63150/63150fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Open the neck skin with a sterile scalpel, from the chin to the upper edge of the breastbone (1.5 cm), and bluntly separate salivary glands from their surrounding connective tissue.</li>
	<li>Separate the muscle group along one side [in this case, the right side] of the trachea, exposing the common carotid artery (CCA) sheath covered with nourishing blood vessels and venules. The CCA and the vagal nerve are located in close proximity to each other.</li>
	<li>Dissociate the CCA and leave a free 8-0 silk suture around the CCA without ligating it in advance. Pay attention to the protection of the vagal nerve, as it is easily damaged (Figure 1A).</li>
	<li>A triple bifurcation of the CCA, the ICA, and the ECA is visible along the lower posterior third of the diastasis. Dissect the distal end of the ECA and ligate the vessel twice as far distaally as possible.</li>
	<li>Disconnect the ECA at the midpoint of the twice ligated segment, creating a vessel stump.</li>
	<li>Prearrange one ligation for the filament around the ECA stump, do not close it until successful filament insertion.</li>
	<li>Use a suture or micro clip to occlude the ICA and CCA temporarily (Figure 1B).</li>
	<li>Make a small incision (approximately half of the ECA diameter) in the ECA using microvascular scissors. Insert a 5-0 (alternatively 4-0) prolene filament into the ECA and advance it into the CCA.</li>
	<li>Close the ligature on the ECA slightly while loosening the micro clip on the ICA and CCA (Figure 1C).</li>
	<li>Gently pull back on the filament and adjust the ECA stump in the cranial direction, invaginating the filament through the bifurcation into the ICA (Figure 1D).</li>
	<li>Point the filament tip medially at an angle of ~30&#176; to the tracheal midline and ~30&#176; to the horizontal plane. Push the filament forward inside the ICA. After reaching the ACA-MCA bifurcation, resistance is encountered (~9 mm).</li>
	<li>Advance the filament 3 mm further, perforating the right ACA. Promptly withdraw the filament to the ECA stump, allowing blood flow into the subarachnoid space.</li>
	<li>Keep the filament in this position for about 10 s (Figure 1E). The presence of muscle tremors, ipsilateral miosis, gasping for breath, altered heart rhythm, and urinary incontinence can be supporting evidence of successful surgery.</li>
	<li>Temporarily close the CCA to avoid excess blood loss. Pull out the filament instantly and ligate the ECA with the prearranged suture. Reopen the CCA and allow reperfusion and further effusion of blood into the subarachnoid space (Figure 1F).</li>
	<li>After checking for bleeding leakage, disinfect the skin surrounding the wound to prevent postoperative skin infections, and suture the wound with a non-absorbable 4-0 polyester fiber suture.</li>
	<li>Place the mouse in a thermal box until consciousness is regained. Wait until the animal is fully awake and ensure it has regained sufficient consciousness to maintain sternal recumbency. Do not return animals to the company of other mice until fully recovered.</li>
	<li>Administer 200-300 mg/kg body weight&#160;paracetamol for postoperative pain relief.</li>
	<li>Check on the mice daily after surgery.</li>
</ol>
3. MRI measurement
<ol>
	<li>24 h after surgery, perform MRI using a rodent scanner (Table of Materials) and a dedicated mouse head resonator- here, a 20 mm transmit/receive quadrature volume resonator was used.</li>
	<li>Place the mouse on a heated circulating water blanket to ensure a constant body temperature of ~37 &#176;C. Induce anesthesia with 2.5 % isoflurane in an O2/N2O mixture (30%/70%) and maintain with 1.5-2 % isoflurane via facemask under continuous ventilation monitoring.</li>
	<li>First perform a fast reference scan acquiring 3 orthogonal slice packages (Tri-Pilot-Multi, FLASH with repetition time TR/echo time TE = 200 ms/3 ms, 1 average, flip angle FA = 30&#176;, field of view FOV = 28 mm x 28 mm, matrix MTX = 256 x 256, slice thickness 1 mm, total acquisition time TA =30 s).</li>
	<li>Then use a high resolution T2-weighted 2D turbo spin-echo sequence for imaging (imaging parameters TR/TE = 5505 ms/36 ms, RARE factor 8, 6 averages, 46 contiguous axial slices with a slice thickness of 0.35 mm to cover the whole brain, FOV = 25.6 mm x 25.6 mm, MTX = 256 x 256, TA = 13 min).</li>
	<li>If the result is unclear, use an additional respiration triggered T2*weighted gradient echo sequence with the same isodistance as the T2w scan (2D FLASH, TR/TE = 600 ms/6.3 ms, FA = 30&#176;, 1 average, 20 axial slices with 0.35 mm thickness, FOV and MTX identical to T2w, TA = 5-10 min depending on the respiration rate).</li>
	<li>Transfer the data into the DICOM image format and use ImageJ software for SAH grading and volumetry of blood clots. Details on the quantification are listed as a step-by-step guide in the supplementary material (Supplementary Figure 1).</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+hemorrhage.">Subarachnoid hemorrhage.</a> Emergency Medicine Clinics of North America. 34 (4), 901-916 (2016).</li><li>Schertz, M., Mehdaoui, H., Hamlat, A., Piotin, M., Banydeen, R., Mejdoubi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+mortality+of+spontaneous+subarachnoid+hemorrhage+in+martinique.">Incidence and mortality of spontaneous subarachnoid hemorrhage in martinique.</a> PLOS ONE. 11 (5), 0155945 (2016).</li><li>Okazaki, T., Kuroda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneurysmal+subarachnoid+hemorrhage:+intensive+care+for+improving+neurological+outcome.">Aneurysmal subarachnoid hemorrhage: intensive care for improving neurological outcome.</a> Journal of Intensive Care. 6 (1), 28 (2018).</li><li>Kilbourn, K. 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No conflicts of interest

In summary, a standardized SAH mouse model induced by endovascular filament perforation operation is presented with minor invasion, short operative time, and acceptable mortality rates. MRI is conducted 24 h postoperatively to ensure the correct bleeding site and the exclusion of other relevant intracranial pathologies. Furthermore, we classified different SAH bleeding grades and measured bleeding volumes, allowing further subgroup analyses based on bleeding grade.
Adequate positioning of the ...

Subarachnoid hemorrhage (SAH) is caused by the rupture of an intracranial aneurysm and poses a life-threatening emergency, associated with substantial morbidity and mortality, accounting for approx. 5% of strokes1,2. SAH patients present with severe headaches, neurological dysfunction, and progressive disturbance of consciousness3. Around 30% of SAH patients die within the first 30 days after the initial bleeding event4. Clinically, 50% of patients experience delayed brain injury (DBI) after early brain injury. DBI is characterized by delayed cerebral ischemia and delayed neurological deficits. Current studies have shown that the synergistic effects of several different factors lead to the loss of neurological function, including the destruction of the blood-brain barrier, the contraction of small arteries, microcirculatory dysfunction, and thrombosis5,6.
One unique aspect of SAH is that the pathogenesis originates from an extraparenchymal location but then leads to detrimental cascades inside the parenchyma: the pathology begins with the accumulation of blood in the subarachnoid space, triggering a multitude of intraparenchymal effects, such as neuroinflammation, neuronal and endothelial cell apoptosis, cortical spreading depolarization, and brain edema formation7,8.
Clinical research is limited by several factors, making the animal model a critical element in consistently and accurately mimicking the pathomechanistic changes of the disease. Different SAH model protocols have been proposed, e.g., autologous blood injection into the cisterna magna (ACM). Also, a modified method with a double injection of autologous blood into the cisterna magna and optic chiasm cistern (APC) respectively9,10. While autologous blood injection is a simple way to simulate the pathological process of vasospasm and inflammatory reactions after subarachnoid hemorrhage, the following rise of intracranial pressure (ICP) is relatively slow, and no noteworthy changes in the permeability of the blood-brain barrier are induced11,12. Another method, the periarterial blood placement, usually used in large SAH models (e.g., monkeys and dogs), involves placing anticoagulated autologous blood or comparable blood products around the vessel. The diameter changes of the artery can be observed with a microscope, serving as an indicator for cerebral vasospasm after SAH13.
Barry et al. first described an endovascular perforation model in 1979 in which the basilar artery is exposed after removing the skull; the artery is then punctured with tungsten microelectrodes, using a microscopic stereotactic technique14. In 1995, Bederson and Veelken modified the Zea-Longa model of cerebral ischemia and established the endovascular perforation, which has been continuously improved ever since15,16. This method is based on the fact that mice and humans share a similar intracranial vascular network, known as the circle of Willis.
For postoperative evaluation and grading of SAH in the mouse model, different approaches have been proposed. Sugawara et al. developed a grading scale that has been widely used since 200817. This method assesses the severity of SAH based on morphological changes. However, for this method, the mouse&#39;s brain tissue morphology must be examined under direct vision, and therefore, the mouse must be sacrificed for assessment. Furthermore, several methods for determining SAH severity in vivo have been established. Approaches range from simple neurological scoring to monitoring of intracranial pressure (ICP) to various radiological imaging techniques. Furthermore, MRI grading has been shown as a new, non-invasive tool to grade SAH severity, correlating to neurological score18,19.
Here, a protocol for an SAH model caused by endovascular perforation is presented, combined with postoperative MRI. In an attempt to establish a system to objectify the amount of bleeding in an in vivo setting, we also developed a system for SAH grading and quantification of total blood volume based on 7.0 T high-resolution T2-weighted MRI. This approach ensures the correct induction of SAH and exclusion of other pathologies such as stroke, hydrocephalus, or intracerebral hemorrhage (ICH) and complications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Eye cream</td>
			<td>Bayer</td>
			<td>815529836</td>
			<td>Bepanthen</td>
		</tr>
		<tr>
			<td>Images analysis software</td>
			<td>ImageJ</td>
			<td></td>
			<td>Bundled with Java 1.8.0_172</td>
		</tr>
		<tr>
			<td>Ligation suture (5-0)</td>
			<td>SMI</td>
			<td></td>
			<td>Silk black USP</td>
		</tr>
		<tr>
			<td>Light source for microscope</td>
			<td>Zeiss</td>
			<td>CL 6000 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>CP-pharma</td>
			<td>797-037</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>MRI</td>
			<td>Bruker</td>
			<td>Pharmascan 70/16</td>
			<td>&#160;7 Tesla</td>
		</tr>
		<tr>
			<td>MRI images acquired software</td>
			<td>Bruker</td>
			<td></td>
			<td>Bruker Paravision 5.1</td>
		</tr>
		<tr>
			<td>Paracetamol (40 mg/mL)</td>
			<td>bene Arzneimittel</td>
			<td>4993736</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolene filament (5-0)</td>
			<td>Erhicon</td>
			<td>EH7255</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Wella</td>
			<td>HS61</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (Fine Scissors)</td>
			<td>FST</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (forceps#1)</td>
			<td>AESCULAP</td>
			<td>FM001R</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (forceps#2)</td>
			<td>AESCULAP</td>
			<td>FD2855R</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (forceps#3)</td>
			<td>Hammacher</td>
			<td>HCS 082-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (Needle holder)</td>
			<td>FST</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (Vannas Spring Scissors)</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilation monitoring</td>
			<td>Stony Brook</td>
			<td></td>
			<td>Small Animal Monitoring &#38; Gating System</td>
		</tr>
		<tr>
			<td>Wounding suture(4-0)</td>
			<td>Erhicon</td>
			<td>CB84D</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylavet</td>
			<td>CP-pharma</td>
			<td>797-062</td>
			<td>20 mg/mL</td>
		</tr>
	</tbody>
</table>

endovascular perforation model for subarachnoid hemorrhage combined with magnetic resonance imaging (mri)

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

Mortality 
For this study, a total of 92 male C57Bl/6J mice aged between 8-12 weeks were subjected to SAH operation; in these, we observed an overall mortality rate of 11.9% (n = 12). Mortality occurred exclusively within the first 6-24 h after surgery, suggesting perioperative mortality as well as SAH bleeding itself as the most likely contributing factors.
SAH bleeding grade 
A total of 50 mice received MRI 24 h postoperatively to confirm SAH and e...

Watch this Scientific Journal Video about Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI at JoVE.com

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI

Here we present a standardized SAH mouse model, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI)

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high ...

endovascular-perforation-model-for-subarachnoid-hemorrhage-combined

Research

JoVE Journal

Neuroscience

3.5K Views.  Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, and Humboldt-Universität zu Berlin, and Berlin Institute of Health. Here we present a standardized SAH mouse model, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies.

Video: Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

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 ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of structural changes caused by genetic or environmental perturbations. Whole-brain imaging results in more accurate quantification of cells and the study of region-specific differences that may be missed with commonly used microscopy of physically sectioned tissue. Using light-sheet microscopy to image cleared brains greatly increases acquisition speed as compared to confocal microscopy. Although these images produce very large amounts of brain structural data, most computational tools that perform feature quantification in images of cleared tissue are limited to counting sparse cell populations, rather than all nuclei.
Here, we demonstrate NuMorph (Nuclear-Based Morphometry), a group of analysis tools, to quantify all nuclei and nuclear markers within annotated regions of a postnatal day 4 (P4) mouse brain after clearing and imaging on a light-sheet microscope. We describe magnetic resonance imaging (MRI) to measure brain volume prior to shrinkage caused by tissue clearing dehydration steps, tissue clearing using the iDISCO+ method, including immunolabeling, followed by light-sheet microscopy using a commercially available platform to image mouse brains at cellular resolution. We then demonstrate this image analysis pipeline using NuMorph, which is used to correct intensity differences, stitch image tiles, align multiple channels, count nuclei, and annotate brain regions through registration to publicly available atlases.
We designed this approach using publicly available protocols and software, allowing any researcher with the necessary microscope and computational resources to perform these techniques. These tissue clearing, imaging, and computational tools allow measurement and quantification of the three-dimensional (3D) organization of cell-types in the cortex and should be widely applicable to any wild-type/knockout mouse study design.

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Tissue clearing followed by light-sheet microscopy (LSFM) enables cellular-resolution imaging of intact brain structure, allowing quantitative analysis of ...

Magnetic Resonance-Guided Stereotaxy for Infusions to the Pig Brain

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide precise infusions. The subject was positioned prone in the MR bore for optimal access to the top of the skull with the torso raised, the neck flexed, and the head inclined downward. Two anchor pins anchored on the bilateral zygoma held the head steady using the head holder. A magnetic resonance imaging (MRI) flex-coil was placed rostrally across the head holder so that the skull was accessible for the intervention procedure. A planning grid placed on the scalp was used to determine the appropriate entry point of the cannula. The stereotactic frame was secured and aligned iteratively through software projection until the projected radial error was less than 0.5 mm. A hand drill was used to create a burr hole for insertion of the cannula. A gadolinium-enhanced co-infusion was used to visualize the infusion of a cell suspension. Repeated T1-weighted MRI scans were registered in real time during the agent delivery process to visualize the volume of gadolinium distribution. MRI-guided stereotaxy allows for precise and controlled infusion into the pig brain, with concurrent monitoring of cannula insertion accuracy and determination of the agent volume of distribution.

The protocol presented here demonstrates stereotaxy to the pig brain using convection-enhanced infusions, with real-time magnetic resonance imaging (MRI) visualization guidance and real-time infusion distribution visualization.

The overall goal of this procedure is to perform stereotaxy in the pig brain with real-time magnetic resonance (MR) visualization guidance to provide ...