The authors would like to thank Jules Morin for insightful comments on the manuscript. This research was funded by awards from the CADASIL Together We Have Hope non-profit organization, the Center for Women&#39;s Health and Research, and the NHLBI R01HL136636 (FD).

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado, Anschutz Medical Campus and were performed according to the guidelines from the National Institutes of Health.
1. Solutions
<ol>
	<li>Use MOPS-buffered saline for the dissection and to keep samples at 4 &#176;C before their utilization. Do not gas the solution. Prepare MOPS buffered saline with following composition: 135 mM NaCl, 5 mM KCl, 1 mM KH2PO4,...

<ol>
	<li>Nishimura, N., Schaffer, C. B., Friedman, B., Lyden, P. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrating+arterioles+are+a+bottleneck+in+the+perfusion+of+neocortex.">Penetrating arterioles are a bottleneck in the perfusion of neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (1), 365-370 (2007).</li><li>Shih, A. Y., 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=Robust+and+fragile+aspects+of+cortical+blood+flow+in+relation+to+the+underlying+angioarchitecture.">Robust and fragile aspects of cortical blood flow in relation to the underlying angioarchitecture.</a> Microcirculation (New York, N.Y.:1994). 22 (3), 204-218 (2015).</li><li>Cipolla, M. J., Smith, J., Kohlmeyer, M. M., Godfrey, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKCa+and+IKCa+Channels,+Myogenic+Tone,+and+Vasodilator+Responses+in+Middle+Cerebral+Arteries+and+Parenchymal+Arterioles:+Effect+of+Ischemia+and+Reperfusion.">SKCa and IKCa Channels, Myogenic Tone, and Vasodilator Responses in Middle Cerebral Arteries and Parenchymal Arterioles: Effect of Ischemia and Reperfusion.</a> Stroke. 40 (4), 1451-1457 (2009).</li><li>Nystoriak, M. A., 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=Fundamental+increase+in+pressure-dependent+constriction+of+brain+parenchymal+arterioles+from+subarachnoid+hemorrhage+model+rats+due+to+membrane+depolarization.">Fundamental increase in pressure-dependent constriction of brain parenchymal arterioles from subarachnoid hemorrhage model rats due to membrane depolarization.</a> AJP: Heart and Circulatory Physiology. 300 (3), H803-H812 (2011).</li><li>Dabertrand, F., Nelson, M. T., Brayden, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acidosis+dilates+brain+parenchymal+arterioles+by+conversion+of+calcium+waves+to+sparks+to+activate+BK+channels.">Acidosis dilates brain parenchymal arterioles by conversion of calcium waves to sparks to activate BK channels.</a> Circulation Research. 110 (2), 285-294 (2012).</li><li>Dabertrand, F., Nelson, M. T., Brayden, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ryanodine+receptors,+calcium+signaling,+and+regulation+of+vascular+tone+in+the+cerebral+parenchymal+microcirculation.">Ryanodine receptors, calcium signaling, and regulation of vascular tone in the cerebral parenchymal microcirculation.</a> Microcirculation (New York, N.Y.:1994). 20 (4), 307-316 (2013).</li><li>Cipolla, M. J., 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=Increased+pressure-induced+tone+in+rat+parenchymal+arterioles+vs.+middle+cerebral+arteries:+role+of+ion+channels+and+calcium+sensitivity.">Increased pressure-induced tone in rat parenchymal arterioles vs. middle cerebral arteries: role of ion channels and calcium sensitivity.</a> Journal of Applied Physiology. 117 (1), 53-59 (2014).</li><li>De Silva, T. M., Modrick, M. L., Dabertrand, F., Faraci, F. 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Y., 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=The+smallest+stroke:+occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit.">The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nature Neuroscience. 16 (1), 55-63 (2013).</li><li>Koide, 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=The+yin+and+yang+of+KV+channels+in+cerebral+small+vessel+pathologies.">The yin and yang of KV channels in cerebral small vessel pathologies.</a> Microcirculation (New York, N.Y.:1994). 25 (1), (2018).</li><li>Girouard, H., 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=Astrocytic+endfoot+Ca2++and+BK+channels+determine+both+arteriolar+dilation+and+constriction.">Astrocytic endfoot Ca2+ and BK channels determine both arteriolar dilation and constriction.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3811-3816 (2010).</li><li>Dabertrand, F., 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=Prostaglandin+E2,+a+postulated+astrocyte-derived+neurovascular+coupling+agent,+constricts+rather+than+dilates+parenchymal+arterioles.">Prostaglandin E2, a postulated astrocyte-derived neurovascular coupling agent, constricts rather than dilates parenchymal arterioles.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 33 (4), 479-482 (2013).</li><li>Longden, T. A., Dabertrand, F., Hill-Eubanks, D. C., Hammack, S. E., Nelson, M. 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W., Sullivan, M. N., Pritchard, H. A. T., Robinson, J. J., Earley, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unitary+TRPV3+channel+Ca2++influx+events+elicit+endothelium-dependent+dilation+of+cerebral+parenchymal+arterioles.">Unitary TRPV3 channel Ca2+ influx events elicit endothelium-dependent dilation of cerebral parenchymal arterioles.</a> AJP: Heart and Circulatory Physiology. 309 (12), H2031-H2041 (2015).</li><li>Johnson, A. C., Cipolla, M. 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The pressurized HiCaPA (hippocampal capillary-parenchymal arteriole) preparation described in the present manuscript is an extension of our well-established procedure to isolate, pressurize, and study parenchymal arterioles29. We recently reported that Kir2.1 channels in brain capillary endothelial cells sense increases in [K+]o associated with neural activation, and generate an ascending hyperpolarizing signal that dilates upstream arterioles20. Revea...

The pial circulation on the surface of the brain has been the object of much study, partly because of its experimental accessibility. However, the topology of the cerebral vasculature creates distinct regions. In contrast to the robust pial network rich in anastomoses with substantial capacity for redirecting the blood flow, the intracerebral parenchymal arterioles (PAs) present limited collateral supply, each of them perfusing a discrete volume of nervous tissue1,2. This creates a bottleneck effect on the blood flow which, combined with unique physiological features3,4,5,6,7,8, makes intracerebral arterioles a crucial site for cerebral blood flow (CBF) regulation9,10. Despite the technical challenges inherent to the isolation and cannulation of PAs, the last decade has seen an increased interest in ex vivo functional studies using pressurized vessels11,12,13,14,15,16,17. One of the reasons for this increased interest is the considerable research effort conducted on neurovascular coupling (NVC), the mechanism sustaining the brain functional hyperemia18.
Regionally, CBF can rapidly increase following local neural activation19. The cellular mechanisms and signaling properties controlling NVC are incompletely understood. However, we identified a previously unanticipated role for the brain capillaries during NVC in sensing neural activity and translating it into a hyperpolarizing electrical signal to dilate upstream arterioles20,21,22. Action potentials23,24 and opening of large-conductance Ca2+-activated K+ (BK) channels on the astrocytic endfeet25,26 increase the interstitial potassium ion concentration [K+]o, which results in activation of strong inward rectifier K+ (Kir) channels in the vascular endothelium of capillaries. This channel is activated by external K+ but also by hyperpolarization itself. Spreading through gap junctions, the hyperpolarizing current then regenerates in adjacent capillary endothelial cells up to the arteriole, where it causes myocyte relaxation and CBF increase20,21. The study of this mechanism led us to develop a pressurized capillary-parenchymal arteriole (CaPA) preparation to measure the arteriolar diameter during capillary stimulation with vasoactive agents. The CaPA preparation is composed of a cannulated intracerebral arteriole segment with an intact, downstream capillary ramification. The capillary ends are compressed against the chamber glass bottom by a micropipette, which occludes and stabilizes the entire vascular formation20,21.
We previously made instrumental innovations by imaging CaPA preparations from the mouse cortex20,21 and arterioles from the rat amygdala13 and hippocampus16,17. As the hippocampal vasculature receives more attention due to its susceptibility to pathological conditions, here we provide a step-by-step method for CaPA preparation from the mouse hippocampus (HiCaPA) that can not only be used in functional NVC studies but also in molecular biology, immunochemistry, and electrophysiology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22&#181;m Syringe Filters</td>
			<td>CELLTREAT Scientific Products</td>
			<td>229751</td>
			<td></td>
		</tr>
		<tr>
			<td>12-0 Nylon (12cm) Black</td>
			<td>Microsurgery Instruments, Inc</td>
			<td>S12-0 NYLON</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm</td>
			<td>Sutter Instruments</td>
			<td>B120-69-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Scope</td>
			<td>Olympus</td>
			<td>SZ11</td>
			<td></td>
		</tr>
		<tr>
			<td>ECOLINE VC-MS/CA 4-12 &#8212; complete Pump with Drive and MS/CA 4-12 pump-head</td>
			<td>Ismatec</td>
			<td>ISM 1090</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14063-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Water Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Integra&#8482; Miltex&#8482;Tissue Forceps</td>
			<td>Fisher Scientific</td>
			<td>12-460-117</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5379</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M1880</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td></td>
		</tr>
		<tr>
			<td>ML 133 hydrochloride</td>
			<td>Tocris</td>
			<td>4549</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr>
			<td>NS309</td>
			<td>Tocris</td>
			<td>3895</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer III - Intracellular Microinjection Dispense Systems, 2-channel</td>
			<td>Parker Hannifin</td>
			<td>052-0500-900</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Servo Controller with Peristaltic Pump</td>
			<td>Living Systems Instrumentation</td>
			<td>PS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P3662</td>
			<td></td>
		</tr>
		<tr>
			<td>Super Fine Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp-Blunt</td>
			<td>Fine Science Tools</td>
			<td>14001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Micropipette Puller</td>
			<td>Narishige</td>
			<td>PP-83</td>
			<td></td>
		</tr>
	</tbody>
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ex vivo pressurized hippocampal capillary-parenchymal arteriole preparation for functional study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

Endothelial small-conductance (SK) and intermediate-conductance (IK) Ca2+-sensitive K+ channels exert a dilatory influence on the diameter of PAs. Bath application of 1 &#181;M NS309, a synthetic IK and SK channel agonist, caused near maximal dilation (Figure 2A,B). However, capillary endothelial cells lack IK and SK channels and did not hyperpolarize in response to NS30920. As a result, stimulati...

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Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

Our method allows the study of blood vessels located in structures that would otherwise be difficult to image in Vivo, such as the hippocampus. Because the blood vessels are removed from the brain tissue, the signaling between the capillaries and the arteriole can be studied without off-target effects on the surrounding tissue. To isolate the hippocampus, use small dissection scissors to cut the skin along the midline at the top of the head of the mouse and move the skin to the sides.Starting at the caudal side of the skull, cut the skull along the midline until the olfactory bulbs are reached and remove portions of the skull until the cerebrum is exposed. Starting near the nose of the mouse, slowly remove the brain, using the scissors to cut through the olfactory bulbs, cranial nerves, and spinal cord. Submerge the brain, ventral side down in MOPS Buffered Saline, in the center of a dissection plate, and place the plate under a dissection microscope.Holding the sharp edge parallel to the bottom of the dish, use razor blade to cut the brain in half along the longitudinal fissure in one stroke. Place one hemisphere in the center of the plate with the midline facing down and push the razor blade straight through the tissue along the transverse fissure to remove the cerebellum and brain stem. Rotate the hemisphere so that the medial side is facing up and use a spatula to hold the brain in place.Insert the tip of a second spatula below the corpus callosum, scooping under the tissue to remove the thalamus, septum, and hypothalamus from the hippocampus. The hippocampus should now be visible as a curved structure near the posterior side of the cerebrum. Using one spatula to hold the cerebrum in place, use the second spatula to scoop the hippocampus out of the cerebrum.Then transfer the hippocampi to a new dissection plate, filled about halfway with fresh MOP solution. For hippocampal arteriole isolation, place small pins at each end of one hippocampus to secure the sample hippocampal artery side up. Using very sharp forceps, gently stretch small sections of the hippocampi to loosen the tissue surrounding the arterioles.And search through the dorsal hippocampal tissues to identify the external transverse arteries. When the artery has been located, grab the external transverse artery from the hippocampus and slowly pull it away from the tissue to collect the arterioles and capillaries. The artery from each hippocampus is removed separately.When there are no more vessels to be removed, place the samples on ice and discard the remaining hippocampal tissue. For cannulation of the arterioles, locate and arteriole with a branch that ends with capillaries and place the arteriole into an organ chamber. Carefully push the cannula tip through the arteriole wall below the target area to mount the blood vessel.Then, carefully slide the vessel onto the cannula until there is enough tissue on which to place the tie. Use 12-0 nylon sutures to make a loose knot that will fit over the blood vessel and cannula, and use a half hitch knot to secure the ties, pull the ends to tighten the knot to secure the arteriole to the cannula. Secure another tie on the other end of the arteriole to seal it.On the opposite side of the chamber, lower a second cannula until the point of the cannula pins down the tie on the end of the arteriole. Then use a third cannula to pin the capillary branch to the coverslip, placing the tip close to the end of the branch, while leaving the ends of the capillaries exposed. As the brain microcirculation is exquisitely fragile, be sure to minimize the stretching and handling of the vessels during the cannulation procedure to ensure survival of the arterioles.For pressure myography analysis, transfer the chamber to a light microscope stage with recording software and connect the inflow and outflow tubing to the chamber for profusion. Start the profusion with 37 degrees Celsius Artificial cerebrospinal fluid at a four milliliter per minute flow rate, and attach the pressuring cannula to peristaltic pump paired with a pressure transducer. Bring the internal pressure to 20 millimeters of Mercury and start the recording software.Adjust the microscope and the image settings to achieve the clearest image possible. And use the Edge Detection software to check that the arteriole is about 15 to 30 micrometers in diameter when it is fully dilated. Once the settings have been optimized, begin the recording and increase the intravascular pressure in the vessel up to 40 millimeters of Mercury, while recording the arteriole diameter with the Edge Detection software.Then profuse the chamber for 15 to 20 minutes to wash out the MOP solution. To test the viability of the vessel, apply one micromolar NS309 solution to the bath profusion, the arteriolar segment should dilate, demonstrating a 30 to 40%myogenic tone. Once the baseline tone for the arteriole has been established, use a glass puller to make cannulas with a fine point at one end, and use forceps to break the tip of each cannula, so that the drug of interest can flow smoothly through the tip at five pounds of pressure per square inch.Fill the cannula with the drug of interest. And load the cannula onto a three-axis micromanipulator attached to the microscope. Connect the tubing from the pressure ejection system to the cannula, and slowly lower the cannula into the bath near the capillaries, taking care not to hit any part of the vessel or the chamber.To stimulate the capillaries, lower the cannula to the coverslip just next to the capillaries and activate the pressure ejection system with the desired ejection time. At the end of the stimulation, raise the cannula slightly to avoid further stimulation. To confirm that only the capillaries were being stimulated, fill the new cannula with one micromolar of NS309 solution and stimulate the capillaries as demonstrated.Then obtain the maximal vasodilation by bathing the preparation with the Calcium-free solution. Bath application of a one micromolar NS309 causes a near maximal dilation of the arteriole due to the Calcium-sensitive Potassium channels in the endothelium. Capillary endothelial cells however, lack intermediate and small conductance channels and do not hyperpolarize in response to the agonist, as a result, stimulating capillary ends with NS309, does not cause upstream arteriolar dilation, this indicates that NS309 did not reach the arteriole, and can be used as a control to access the spacial restriction of the compound applied onto capillaries by pressure ejection.Using the hippocampal capillary parenchymal arteriole preparation as demonstrated, the application of a Artificial cerebrospinal fluid supplemented with 10 millimolar Potassium to the capillary ends, results in an upstream arteriolar dilation that does not differ between preparations from male and female mice. Further, the addition of the Kir2 inhibitor ML 133, virtually abolishes the capillary induced arteriolar dilation, in response to 10 millimolar Potassium in preparations from both male and female mice. When mounting the blood vesssel, limit direct interactions with the blood vessel to minimize the damage to, and increase the viability of the vessel.Once the blood vessel has been isolated, different drug compounds can be tested or the blood vessel can be further processed for olecular biology, immunochemistry, or electrophysiology studies.

ex-vivo-pressurized-hippocampal-capillary-parenchymal-arteriole

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JoVE Journal

Neuroscience

6.7K visualizações.  University of Colorado Anschutz Medical Campus. Nosso método permite o estudo de vasos sanguíneos localizados em estruturas que de outra forma seriam difíceis de imaginar na Vivo, como o hipocampo. Como os vasos sanguíneos são removidos do tecido cerebral, a sinalização entre os capilares e a arteriola pode ser estudada sem efeitos fora do alvo no tecido circundante. Para isolar o hipocampo, use uma pequena tesoura de dissecção para cortar a pele ao longo da linha média na parte superior da cabeça do rato e mover a pele para os ...