Name: in vivo fiber-coupled pre-clinical confocal laser-scanning endomicroscopy (pcle) of hippocampal capillaries in awake mice
Uploaded: 2023-04-21T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice at JoVE.com

The project was funded by&#160;a Research Initiative Award from the American Epilepsy Society, and an award from the Arizona Biomedical Research Commission to S.L.M., as well as a challenge grant from Research to Prevent Blindness Inc. to the Department of Ophthalmology at SUNY Downstate Health Sciences University, the New York State Empire Innovation Program, and further grants from the National Science Foundation (0726113, 0852636, &#38; 1523614), the Barrow Neurological Foundation, Mrs. Marian Rochelle, Mrs. Grace Welton, and Dignity Health SEED awards, and by federal grants from the National Science Foundation (0726113, 0852636, &#38; 1523614) and by the National Institute of Health (Awards R01EY031971 AND R01CA258021), to S.L.M and S.M.C.&#160;<span lang="EN-US" style="font-size: 12pt; line-height: 107%; font-family: Calibri, sans-serif; color: rgb(34, 34, 34); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">This work was also supported by the Office of the Assistant Secretary of Defense for Health Affairs under Award No. W81XWH-15-1-0138, to S.L.M..<span lang="EN-US" style="font-size: 12pt; line-height: 107%; font-family: Calibri, sans-serif; color: red; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;<span lang="EN-US" style="font-size: 12pt; line-height: 107%; font-family: Calibri, sans-serif; color: rgb(34, 34, 34); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">L.-C. was supported by a Jos&#233; Castillejo fellowship from the Spanish Ministry of Education. We thank O. Caballero and M. Ledo for their technical advice and assistance.&#160;

The protocol follows the NIH Guidelines for the Care and Use of Laboratory Animals. All procedures were approved by the Barrow Neurological Institute&#8217;s Institutional Animal Care and Use Committee.
1. Stereotaxic positioning for craniotomy
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	<li>Weigh and then anesthetize the mouse with a ketamine-xylazine (100 mg/kg&#8211;10 mg/kg i.p.) cocktail. Ensure that the animal is fully anesthetized by observing its lack of reaction to tail and/or toe pinch.</li>
	<li>Place a heating pad un...

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We developed a head-cap restraint system for simultaneous electrophysiological and fiber-optic pCLE experiments in awake mice, reducing potential response contamination due to anesthetic drugs. The head-cap and mounting apparatus are straightforward to construct and are reusable for chronic awake-behaving imaging experiments. We checked the quality of the recordings against the gold-standard for in vivo microscopic blood flow imaging, TPLSM.
Proficient surgical skills are necessary to implemen...

In contrast with other microscopic imaging methods1,2,3,4,5,6,7,8, in vivo fiber-optic-based confocal microscopy allows the measurement of blood flow dynamics in any brain region, at any depth, at high speed (up to 240 Hz depending on field-of-view size9). A fiber-optic probe enables in vivo confocal laser scanning imaging at 3 &#181;m resolution because the tip of the probe (a lens-less objective made up of a bundle of 5000-6000 3 &#181;m diameter individual fibers) can be positioned with a microelectrode&#8217;s accuracy, within 15 &#181;m of the fluorescent target of interest. As with in vivo two-photon imaging, fluorophores must be previously introduced into the imaging target. For example, fluorescein dextran (or quantum dots) may be injected into the vasculature, or genetically-encoded fluorescent proteins can be transfected into cells, or fluorescent dyes such as Oregon Green BAPTA-1 can be bulk-loaded into cells, prior to imaging.
Recent research using these techniques has found that mural cell motor activity leading to ictal capillary vasospasms&#8212;sudden constrictions that occur at the position of the mural cells during seizures9&#8212;can contribute to neurodegeneration in the ictal hippocampus9. Whereas previous imaging studies showed in vitro and in vivo pericyte constrictions connected to drug applications6,7,10,11,12, Leal-Campanario et al. found the first evidence of in vivo spontaneous capillary constrictions in the murine brain. To establish relevance to human temporal lobe epilepsy, they studied male (P30-40 old) knockout (KO) Kv1.1 (kcna1-null) mice14,15 (JAX stock #003532), a genetic model of human episodic ataxia type 115. Pericytes drove both pathological and physiological hippocampal mural vasoconstrictions9 in the spontaneously epileptic animals and their wild-type (WT) littermates. These observations were replicated in WT animals rendered epileptic with kainic-acid, thereby indicating their generalization to other forms of epilepsy. Leal-Campanario et al moreover determined, using novel stereological microscopy approaches, that apoptotic&#8212;but not healthy&#8212;neurons in epileptic animals were spatially coupled to the hippocampal microvasculature. Because excitotoxicity has no known spatial association to the vasculature, this result indicated that abnormal capillary vasospasmic ischemia-induced hypoxia contributes to neurodegeneration in epilepsy. Figure 1 shows a schematic of the general setup.

<table>
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			<td>0.7 mm diameter burr</td>
			<td>Fine Science Tools</td>
			<td>19007-07</td>
			<td>For Screws No. 19010-00</td>
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			<td>0.9 mm diameter burr</td>
			<td>Fine Science Tools</td>
			<td>19007-09</td>
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			<td>ASEPTICO AEU-12C TORQUE PLUS</td>
			<td>from Handpiece solution</td>
			<td>AEU12C</td>
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			<td>Bull dog serrifine clump</td>
			<td>Fine Science Tools</td>
			<td>18050-28</td>
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			<td>CellVizio dual band</td>
			<td>Mauna Kea Technologies</td>
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			<td>CellVizio single band</td>
			<td>Mauna Kea Technologies</td>
			<td></td>
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			<td>Confocal Microprobe 300 microns (Serie S)</td>
			<td>Mauna Kea Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Custom-made alignment piece</td>
			<td>L-shaped (angled at 90 deg) and made of stainless steel with two holes drilled on it, with a 4 mm separation from center to center</td>
			<td></td>
			<td></td>
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			<td>Custom-made mounting bar</td>
			<td>The long section piece of the mounting bar should be between 9.4 - 13mm. Fixed to this piece of the mounting bar, position a stainless-steel plate 1.5 cm long and 0.5 cm wide that has two holes drilled separated 4 mm from center to center, the same distance that the L-shaped alignment piece.</td>
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			<td>Cyanoacrylate adhesive-Super Glue</td>
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			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
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			<td>DuraLay Inlay Resin &#8211; Standard Package</td>
			<td>Reliance Dental Mfg Co.</td>
			<td>602-7395 (from patterson dental)</td>
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			<td>Fillister Head, Slotted Drive, M1.6x0.35 Metric Coarse, 12mm Length Under Head, Machine Screw</td>
			<td>MSC industrial direct co.</td>
			<td>2834117</td>
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			<td>Fine Point scissor</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
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			<td>Fluorescein 5% w/w lysine-fixable dextran (2MD)</td>
			<td>Invitrogen, USA</td>
			<td>D7137</td>
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			<td>Halsey smooth needle holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
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			<td>Kalt suture needle 3/8 curved</td>
			<td>Fine Science Tools</td>
			<td>12050-03</td>
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			<td>lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting Co. 51704</td>
			<td>51670</td>
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			<td>Methocel 2%</td>
			<td>Omnivision GmbH</td>
			<td>PZN: 04682367</td>
			<td>Eye ointment to prevent dryness.</td>
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			<td>Mouse Temperature controller, probe (YSI-451), small heating pad-TC-1000 Mouse</td>
			<td>CWE Inc.</td>
			<td>08-13000</td>
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			<td>PhysioTel F20-EET transmitters</td>
			<td>DSI</td>
			<td>270-0124-001</td>
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			<td>Robot Stereotaxic, Manipulator Arm, ADD-ON, 3 Axis, LEFT</td>
			<td>Stoelting Co.C13</td>
			<td>51704</td>
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			<td>Sel-Tapping bone screws</td>
			<td>Fine Science Tools</td>
			<td>19010-10</td>
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			<td>Standard Ear Bars and Rubber Tips for Mouse Stereotaxic</td>
			<td>Stoelting Co</td>
			<td>51648</td>
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			<td>Suture Thread - Braided Silk/Size 4/0</td>
			<td>Fine Science Tools</td>
			<td>18020-40</td>
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			<td>Tissue separating microspatula</td>
			<td>Fine Science Tools</td>
			<td>10091-121</td>
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</table>

in vivo fiber-coupled pre-clinical confocal laser-scanning endomicroscopy (pcle) of hippocampal capillaries in awake mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

We developed these methods to assess whether abnormal pericyte-driven capillary vasospasms in the hippocampus&#8212;occurring as the result of seizures&#8212;could cause frank hypoxia that contributes to cell death in the ictal focus9,13.
The development of the head-cap and its proper installation afforded high-stability in the recordings, allowing simultaneous recording of EEG and blood flow deep in the hippocampus of wild-type and ep...

Watch this Scientific Journal Video about In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice at JoVE.com

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

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