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
<ol>
	<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...

<ol>
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S., Tank, D. W., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+miniature+head-mounted+two-photon+microscope.+High-resolution+brain+imaging+in+freely+moving+animals.">A miniature head-mounted two-photon microscope. High-resolution brain imaging in freely moving animals.</a> Neuron. 31, 903 (2001).</li><li>Chaigneau, E., Oheim, M., Audinat, E., Charpak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+capillary+blood+flow+in+olfactory+bulb+glomeruli.">Two-photon imaging of capillary blood flow in olfactory bulb glomeruli.</a> Proceedings of the National Academy of Sciences of the United States of America. 100, 13081-13086 (2003).</li><li>Larson, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-soluble+quantum+dots+for+multiphoton+fluorescence+imaging+in+vivo.">Water-soluble quantum dots for multiphoton fluorescence imaging in vivo.</a> Science. 300, 1434-1436 (2003).</li><li>Hirase, H., Creso, J., Singleton, M., Bartho, P., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+brain+pericytes+in+vivo+using+dextran-conjugated+dyes.">Two-photon imaging of brain pericytes in vivo using dextran-conjugated dyes.</a> Glia. 46, 95-100 (2004).</li><li>Hirase, H., Creso, J., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+level+imaging+of+local+cerebral+blood+flow+in+bicuculline-induced+epileptic+foci.">Capillary level imaging of local cerebral blood flow in bicuculline-induced epileptic foci.</a> Neuroscience. 128, 209-216 (2004).</li><li>Schaffer, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+cortical+surface+microvessels+reveals+a+robust+redistribution+in+blood+flow+after+vascular+occlusion.">Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion.</a> PLoS Biology. 4, 22 (2006).</li><li>Leal-Campanario, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+Capillary+Vasodynamics+Contribute+to+Ictal+Neurodegeneration+in+Epilepsy.">Abnormal Capillary Vasodynamics Contribute to Ictal Neurodegeneration in Epilepsy.</a> Scientific Reports. 7, 43276 (2017).</li><li>Peppiatt, C. M., Howarth, C., Mobbs, P., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+control+of+CNS+capillary+diameter+by+pericytes.">Bidirectional control of CNS capillary diameter by pericytes.</a> Nature. 443, 700-704 (2006).</li><li>Yemisci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+contraction+induced+by+oxidative-nitrative+stress+impairs+capillary+reflow+despite+successful+opening+of+an+occluded+cerebral+artery.">Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery.</a> Nature Medicine. 15, 1031-1037 (2009).</li><li>Fernandez-Klett, F., Offenhauser, N., Dirnagl, U., Priller, J., Lindauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+in+capillaries+are+contractile+in+vivo,+but+arterioles+mediate+functional+hyperemia+in+the+mouse+brain.">Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 22290-22295 (2010).</li><li>Leal-Campanario, R., Alarcon-Martinez, L., Martinez-Conde, S., Calhoun, M., Macknik, S., Mouton, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+Flow+Analysis+in+Epilepsy+Using+a+Novel+Stereological+Approach.">Blood Flow Analysis in Epilepsy Using a Novel Stereological Approach.</a> Neurostereology: Unbiased Stereology of Neural Systems. , (2013).</li><li>Smart, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+of+the+K(V)1.1+potassium+channel+causes+epilepsy+in+mice.">Deletion of the K(V)1.1 potassium channel causes epilepsy in mice.</a> Neuron. 20, 809-819 (1998).</li><li>Zuberi, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mutation+in+the+human+voltage-gated+potassium+channel+gene+(Kv1.1)+associates+with+episodic+ataxia+type+1+and+sometimes+with+partial+epilepsy.">A novel mutation in the human voltage-gated potassium channel gene (Kv1.1) associates with episodic ataxia type 1 and sometimes with partial epilepsy.</a> Brain. 122, 817-825 (1999).</li></ol>

We have nothing to disclose.

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>
	<tbody>
		<tr>
			<td>0.7 mm diameter burr</td>
			<td>Fine Science Tools</td>
			<td>19007-07</td>
			<td>For Screws No. 19010-00</td>
		</tr>
		<tr>
			<td>0.9 mm diameter burr</td>
			<td>Fine Science Tools</td>
			<td>19007-09</td>
			<td></td>
		</tr>
		<tr>
			<td>ASEPTICO AEU-12C TORQUE PLUS</td>
			<td>from Handpiece solution</td>
			<td>AEU12C</td>
			<td></td>
		</tr>
		<tr>
			<td>Bull dog serrifine clump</td>
			<td>Fine Science Tools</td>
			<td>18050-28</td>
			<td></td>
		</tr>
		<tr>
			<td>CellVizio dual band</td>
			<td>Mauna Kea Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellVizio single band</td>
			<td>Mauna Kea Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<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>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive-Super Glue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>DuraLay Inlay Resin &#8211; Standard Package</td>
			<td>Reliance Dental Mfg Co.</td>
			<td>602-7395 (from patterson dental)</td>
			<td></td>
		</tr>
		<tr>
			<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>
			<td></td>
		</tr>
		<tr>
			<td>Fine Point scissor</td>
			<td>Fine Science Tools</td>
			<td>14090-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein 5% w/w lysine-fixable dextran (2MD)</td>
			<td>Invitrogen, USA</td>
			<td>D7137</td>
			<td></td>
		</tr>
		<tr>
			<td>Halsey smooth needle holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalt suture needle 3/8 curved</td>
			<td>Fine Science Tools</td>
			<td>12050-03</td>
			<td></td>
		</tr>
		<tr>
			<td>lab standard stereotaxic, rat and mouse</td>
			<td>Stoelting Co. 51704</td>
			<td>51670</td>
			<td></td>
		</tr>
		<tr>
			<td>Methocel 2%</td>
			<td>Omnivision GmbH</td>
			<td>PZN: 04682367</td>
			<td>Eye ointment to prevent dryness.</td>
		</tr>
		<tr>
			<td>Mouse Temperature controller, probe (YSI-451), small heating pad-TC-1000 Mouse</td>
			<td>CWE Inc.</td>
			<td>08-13000</td>
			<td></td>
		</tr>
		<tr>
			<td>PhysioTel F20-EET transmitters</td>
			<td>DSI</td>
			<td>270-0124-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Robot Stereotaxic, Manipulator Arm, ADD-ON, 3 Axis, LEFT</td>
			<td>Stoelting Co.C13</td>
			<td>51704</td>
			<td></td>
		</tr>
		<tr>
			<td>Sel-Tapping bone screws</td>
			<td>Fine Science Tools</td>
			<td>19010-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Ear Bars and Rubber Tips for Mouse Stereotaxic</td>
			<td>Stoelting Co</td>
			<td>51648</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture Thread - Braided Silk/Size 4/0</td>
			<td>Fine Science Tools</td>
			<td>18020-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue separating microspatula</td>
			<td>Fine Science Tools</td>
			<td>10091-121</td>
			<td></td>
		</tr>
	</tbody>
</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 ...

This protocol allowed us to use the confocal laser-scanning fiber-optic endomicroscope to perform simultaneous imaging and electrophysiological recordings in vivo over several hours from neural structures at any depth of the brain in the awake or relatively anesthetized animal. We developed a painless restraint method that allows for deep brain imaging in awake animals using method like those previously employed for electrophysiological recordings. This method has been used to study functional vascular abnormalities in rodent models of epilepsy and age-related macular degeneration.It is a viable technique to evaluate the contribution of blood flow pathology in many, if not most organs of the diseases. While this technique was developed for a real time study of blood flow dynamics in the brain, we have used it to study blood flow in the in vivo retina. It is not limited to vascular studies in neural tissue.Demonstrating the procedure will be Jose Maria Gonzalez Martin, a highly qualified technician from my lab. After confirming a lack of response to petal reflex, place the teeth inside the hole of the bite bar, and stabilize the head of the mouse in a rodent stereotaxic frame. Tighten the nose clamp until it is snug.The position of the body should be as straight as possible. Use ear bars with jaw holder cuffs to secure the head such that the zygomatic processes of the skull are within the clamp. After cleaning and sterilizing the scalp, make 12 to 15 millimeter incision along the midline from the base of the skull to between the eyes along the sagittal midline of the skull, and use four 28 millimeter Bulldog serrefine clamps to retract the borders of the scalp, exposing the skull, and maximizing the working area.Scrape the periosteum to the incision's edges, and dry the top of the skull. Measure the height of the skull at any distance posterior to bregma, and two equal distances lateral from the midline on either side of the skull. Identify the target point, the right hippocampus with the aid of a stereotaxic atlas.On the skull, find the target point coordinates relative to bregma with the stereotax, and use a surgical marker to mark four corners of a 1.4 by two millimeter imaging window centered around the target point directly above the hippocampus. Use the surgical marker to draw an additional mark over the top left parietal bone, and another over the right frontal bone for the bone screws. Use the marker to draw one one millimeter on each side of the midline, and one dot on the midline one millimeter behind lambda to indicate the epidural EEG recording electrode insertion points.To install the head cap, equip a dental drill with a 0.7 millimeter diameter burr, and carefully drill two small holes in the skull at the bone screw placement points. Next place one 70 to 80%ethanol sterilized four millimeter 0.85 diameter stainless steel slotted bone screw into each hole, taking care that the screw tips do not protrude beyond the bottom of the bone into the skull cavity. Then use an L-shaped custom alignment piece clamped to the microdrive mounted to the stereotaxic device to align the head cap along the midline with the anterior ridge overlying bregma, and insert two Fillister head slotted drive screws over the bregma until the screws lie flat on the skull, taking care not to exert too much pressure on the skull.To set up the imaging window, drill a 0.7 millimeter diameter burr hole at each of the positions of the craniotomy reference marks for the corners of the imaging window before using the drill to gently delineate the periphery of the 1.4 by two millimeter craniotomy window with iteratively deeper cuts. Cover the open window with bone wax, and use a 0.9 millimeter diameter burr to drill the epidural EEG electrode holes, taking care not to cut through the duramater. Cover the three holes with bone wax, and build a wall of dental cement that encompasses the imaging window and EEG holes to bind the wall to the head cap.When the cement has dried, use 4.0 or 5.0 black braided silk and simple interrupted sutures to close any loose wound margins, and use a cotton tipped applicator to apply lidocaine and antibacterial ointments to the exposed skin. Then place the mouse in a warm cage with monitoring until full recovery. One day after the head cap implantation survey, secure the head cap to a custom mounting bar attached to the stereotaxic frame to position the awake mouse securely to the frame throughout the imaging session.Insert the ground epidural EEG recording electrodes into the posterior central hole, and insert the recording electrodes in the anterior left and right holes. The tips of the wires are L-shaped, and positioned between the skull and the duramater. To visualize the blood flow within the hippocampus, secure the tail with two fingers to locate the central vein, and holding an ultra fine insulin syringe equipped with an integrated 30 gauge needle parallel to the tail, insert the needle bevel side up into the vein approximately halfway to two thirds from the base of the tail to deliver 200 microliters of 5%green fluorescein lysine-fixable dextran.Then use the bent tip of a syringe needle to remove the dura and initiate the confocal laser scanning recording. Directly following the tail vein injection, clamp the 300 micron beveled fiber-optic bundle to the mobile arm of the robotic stereotaxic drive in a downward orientation. The restraint system permits stable blood flow recordings of deep brain microvessels, including capillaries and their proximal mural cells over long hours.In this representative analysis of entire microvessels, blood flow stoppages occurred at the labeled mural cells. The quantity of the fiber recordings can be determined by comparing high resolution two photon imaging recordings of the blood flow to equivalent vasospasms within cortical tissue. Immunohistochemical analysis at the recording sites illustrates that hippocampal mural cells are correctly targeted using this method, not only constricting in response to the treatment, but also spatially associating with strictures in microvessels far from arterials.Proficiency in the stereotaxic surgery and lateral tail vein injection is crucial to perform this protocol. Thorough practice is essential to reduce error and improve accuracy. The technique allows for realtime imaging of blood flow dynamics in deep brain structures, making it suitable for studying vascular system pathologies and evaluating the efficacy of treatments that target them.

in-vivo-fiber-coupled-pre-clinical-confocal-laser-scanning

Research

JoVE Journal

Neuroscience

826 visualizações.  Universidad Pablo de Olavide. Este protocolo nos permitiu usar o endomicroscópio confocal de varredura a laser de fibra óptica para realizar imagens simultâneas e registros eletrofisiológicos in vivo durante várias horas a partir de estruturas neurais em qualquer profundidade do cérebro no animal acordado ou relativamente anestesiado. Desenvolvemos um método de contenção indolor que permite imagens cerebrais profundas em animais acordados usando métodos como os anteriormente empregados para registros eletrofisio...