Name: Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation (Video) | JoVE Journal
Uploaded: 2016-01-12T18:00:00.000+00:00
Duration: 10 min 4 s
Description: Learn two-photon microscopy to quantify cerebral vascular architecture in a mouse model of HIV-induced neuroinflammation. This method enables accurate assessment of cortical capillary changes.

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5 minutes

We thank Maria Jepson, Dr. Paivi Jordan, and Dr. Linda Callahan at the University of Rochester Multiphoton Core for technical advice throughout the completion of this protocol. We also thank Dr. Changyong Feng for expert statistical advice, and Dr. Maiken Nedergaard at the University of Rochester Medical Center for the headplate design used in this paper. This work was supported in part by grants T32GM007356 and R01DA026325 from the National Institutes of Health (NIH); and by the University of Rochester Center for AIDS Research grant P30AI078498 (NIH).

The University of Rochester&#39;s University Committee on Animal Resources approved all procedures performed in this paper.
1. Pre-surgical Preparation (and Mice)
<ol>
	<li>Prepare the surgical area with all required equipment. Sterilize all tools used during the procedure beforehand using 70% ethanol. Optionally, use a glass-bead sterilizer or autoclave to sterilize the tools.</li>
	<li>Place the mouse in an isoflurane induction chamber connected to an isoflurane vaporizer. Set isoflurane levels to 4% at a rate of 1 L/min. 
	Note: for the experiments reported here, mice with a doxycycline (DOX) inducible HIV-1 Tat transgene driven by the astrocyte-specific glial fibrillary protein (GFAP) promoter (HIV Tat-Tg mice)10 were used to examine the effect of HIV-1 induced neuroinflammation on cerebral vascular structure, as previously described7,10.</li>
	<li>Gently tip the induction chamber to observe the mouse&#39;s righting reflex. Once the righting reflex has been suppressed, set the isoflurane level to 2% and redirect the airflow from the induction chamber to the nose cone at the surgical bench.</li>
	<li>Quickly move the mouse from the induction chamber to the water infused heating pad. Continue to apply isoflurane (1.5-2%) through the nose cone. Adjust anesthesia according to individual mouse tolerance assessed through respiratory rate (RR) monitoring. 
	Note: The depression of RR can perturb physiologic gas parameters altering cerebral blood flow and vessel diameter assessment. Attempt to keep RR between 55-65 breaths per minute. Heart rate (HR) can also be used assess depth of anesthesia; keep HR between 300-450 beats per minute to prevent physiologic changes to vessel diameter. Typically, anesthesia will be adequate between 1.5-2.0% isoflurane at 1 L/min for a given mouse.</li>
	<li>Perform a toe pinch to further check the depth of anesthesia. If the mouse does not respond, commence the surgical procedures.</li>
</ol>
2. Preparation of the Thin-skull Cranial Window
<ol>
	<li>Apply artificial tear gel to the eyes of the mouse. Completely remove the hair from the scalp of the mouse using scissors or an electric razor.</li>
	<li>Sterilize the scalp by applying povidone-iodine solution; allow to dry. Then, under a light microscope, remove the skin from the scalp of the mouse, completely exposing the parietal bones, caudal frontal bones, and bregma marking.</li>
	<li>Apply a small quantity of a 10% ferric chloride solution to the skull in order to dry the membranes for easy removal. Remove the dried membranes with the #5/45 forceps by gently scraping the skull.</li>
	<li>Apply a thin layer of glue around the headplate window. Gently press the headplate against the skull of the mouse, keeping the area of interest in the center of the window. Apply a drop of dental cement to the headplate to polymerize the glue.</li>
	<li>Apply a thin layer of glue along the edge of the headplate window to create a reservoir in which to hold saline. Once the glue has dried, screw the headplate into the mouse headplate harness. 
	Note: the mouse headplate harness used in this procedure is a structure with a metal base and two metal columns. On each column there is one spring and one wing nut. The headplate is placed on top of the spring, and the wing nut is tightened until the head is level and does not move.</li>
	<li>Remove any glue from the headplate window using a drill bit attached to a microtorque drill set at 6,000 rpm. Stop every 10-15 sec&#160;to prevent overheating of mouse cranium that can result in structural damage to vessels.
	<ol>
		<li>Using a new drill bit, place the microtorque drill 1.5 mm laterally from midline (one-third of the diameter of the headplate window). Begin to thin the skull around this location at 4,000 rpm. Move the drill gently across the skull with no direct downward pressure.</li>
		<li>Cease drilling every 10-15 sec&#160;to prevent overheating the mouse cranium. During this time, remove the accumulated skull dust using a compressed air canister.</li>
		<li>Use drops of RT saline to cool cranium for 5-10 sec. Gently remove all fluid with a soft tissue to continue skull thinning.</li>
	</ol>
	</li>
	<li>For the final thinning of the skull, place a small volume of saline in the headplate reservoir and lightly sweep a dental microblade over the area of interest until small vessels are clearly visible.</li>
</ol>
3. Monitoring of Physiological Parameters
<ol>
	<li>Once the skull is completely thinned, detach the mouse from the holder and place it on its back. Gently tape down both hind legs to clearly expose the medial thighs.</li>
	<li>Remove the hair over both medial thighs with scissors or an electric shaver. Disinfect the surgical site by covering the thighs with povidone-iodine.</li>
	<li>Pinch the skin on the medial right thigh above the femoral vein and artery using the #5 forceps. Gently pull the skin upwards and use scissors to cut and remove the skin. 
	Note: the left thigh is reserved in the event of surgical complications (vascular anomalies, ruptured vessels).</li>
	<li>Apply approximately 3-5 drops of 0.9% saline to surgical site. This allows for greater magnification of vessels, as well as keeping the site moisturized and preventing drying of the vessels. Separate the femoral vein from artery by bluntly dissecting into the femoral neurovascular bundle using two #5/45 forceps.</li>
	<li>Place two, 3 cm pieces of surgical suture underneath the femoral artery approximately 1 cm apart. Twist the upper suture clockwise to create a vascular tourniquet that will help to prevent excessive blood loss during catheterization.</li>
	<li>Make a small incision in the femoral artery using the spring scissors. Place the catheter in the artery through the incision and advance until the catheter is securely within the artery.</li>
	<li>Untwist the upper suture (tourniquet) to assess for leaks and proper catheter placement. Secure the catheter in the femoral artery by tying two knots around the catheter within the vessel using the sutures.</li>
	<li>Inject 10 &#181;l of heparin (100 IU/ml) through the catheter to prevent blood clotting. Then, surgically close the right thigh by sewing the skin together with a 4-0 suture in a simple interrupted pattern.</li>
	<li>Inject 50 &#181;l of urethane (1.2 mg/g) intraperitoneally into the right lower quadrant. Five minutes later repeat with another 50 &#181;l injection to the left lower quadrant for a total of 100 &#181;l of intraperitoneal urethane. Slowly reduce the isoflurane levels to approximately 0.25% while concomitantly rechecking mouse for the depth of anesthesia. 
	Note: Keep the needle superficial within the peritoneal cavity along the posterior abdominal wall so that it does not perforate the bowel. This can be accomplished by pinching the abdominal rectus muscles away from the viscera and then injecting.</li>
	<li>Using a capillary tube, collect a 40 &#181;l sample of arterial blood from the catheter. Attach the catheter to the blood pressure transducer fitted with a saline-filled four-way stopcock and heparin-filled syringe.</li>
	<li>Tape the blood pressure transducer to the surgical apparatus to prevent the unintended removal of the catheter. Begin monitoring of mean arterial pressure.</li>
	<li>Insert the blood filled capillary tube into a blood gas analyzer entry port. Once all the blood has been extracted, remove the capillary tube from the entry port. The O2 and CO2 blood gas levels will appear on a paper printed from the blood gas analyzer.</li>
</ol>
4. Injection of the Fluorescent Dye
<ol>
	<li>Pinch the skin on the medial left thigh using the #5 forceps. Gently pull the skin upwards and use scissors to cut and remove the skin. Prepare dextran conjugated red emitting rhodamine dye (70 kDa) (10 mg/kg dissolved in saline).</li>
	<li>Locate the femoral vein.Using a 1 ml syringe with a 30 G needle attached, draw a previously prepared solution of a fluorescent dye appropriate for imaging to the 130 &#181;l line of the syringe. Remove all air bubbles by drawing the dye completely into the syringe and firmly flicking the syringe wall.
	<ol>
		<li>Bend the 30 G needle at an approximately 30 degree angle by pressing it against a hard surface bevel side up. Fill the needle with the dye and discard any excess liquid, leaving a 100 &#181;l sample.</li>
	</ol>
	</li>
	<li>Inject the 100 &#181;l sample of dye slowly into the femoral vein. After removing the needle, apply steady, gently pressure to the site of injection to stop any bleeding. Allow the dye to circulate for 5 min. 
	Note: alternatively, the right femoral vein can be used instead for the fluorescent dye injection to prevent further blood loss through the creation of another surgical site. In addition, if the animal is bleeding uncontrollably from the surgical site, this may be an indication that too much heparin was given to flush the catheter. If there is no clotting within 10-15 min&#160;and the mouse is still bleeding, consider restarting the experiment with a new mouse.</li>
	<li>Close the left thigh by sewing the skin together with a 4-0 suture, then carefully flip the mouse onto its stomach, and place it into the mouse headplate harness.</li>
</ol>
5. In Vivo Two-Photon Imaging
<ol>
	<li>Move the surgical apparatus to the two-photon microscope, making sure to maintain continuous anesthesia levels. Place a small quantity of 0.9 % saline into the headplate reservoir and lower the microscope objective so that it comes into contact with the saline. Locate the area of interest using the brightfield-viewing objective</li>
	<li>Set the two-photon laser excitation to a wavelength appropriate for the fluorescent dye. Begin two-photon imaging using the 25x objective. 
	Note that in these experiments we used a dextran conjugated to a red emitting rhodamine dye that was excited at a wavelength of 780 nm. A 607/36 bandpass emission filter to detect the fluorescence from the dye, and a 480/20 bandpass emission filter was used to detect arterial autofluorescence</li>
	<li>Locate a capillary bed on the view screen, and magnify this area using optical zoom 2. Acquire images of the capillaries using the two-photon imaging software.</li>
	<li>After imaging is complete, sacrifice the mouse by cervical dislocation followed by decapitation.</li>
</ol>
6. Ex Vivo Two-Photon Imaging
<ol>
	<li>Using the extra fine scissors, make an incision in the scalp from the interparietal bones to the frontal bones of the mouse. Secure the skin to the sides of the skull with the pointer finger and thumb.</li>
	<li>Place the extra fine scissors underneath the medial interparietal bone and cut the skull along the sagittal suture. Stop cutting the skull approximately 3 mm after the bregma marking. 
	Note: Apply upward pressure when cutting the skull to prevent cutting the brain.</li>
	<li>Using the #5 forceps, separate the skull from the brain and carefully remove any meninges from the surface of the brain. Remaining meninges can accidently lacerate the brain; extreme care should be taken to avoid this. Gently slide the #5 forceps under the brain advancing slowly forward until brain is free from skull. 
	Note: that downward pressure should be used in order to avoid puncturing the brain.</li>
	<li>Place the brain in a brain sectioning tool specific for mice. Wash the brain by applying drops of artificial cerebrospinal fluid over the brain.</li>
	<li>Remove a 2 mm coronal section of brain from bregma 0 to bregma -2. Place the coronal section onto a concave glass slide containing artificial cerebrospinal fluid with the 0 bregma (most cranial part of the section) facing upwards. Gently cover the brain slice with a glass cover slip. 
	Note: Avoid pressing the glass once placed on the brain section as this may deform the vascular architecture.</li>
	<li>Transfer the slide to the microscope stage and place a small quantity of 0.9% saline on the cover slip. Lower the microscope objective until it comes in contact with the saline. Locate the midline of the brain using the brightfield objective.</li>
	<li>Begin two-photon imaging and locate the midline again using the 25x objective. Accomplish this by looking for the longitudinal fissure at the cortical surface of the coronal section. Place the right edge of the imaging screen on the midline and move the viewer screen over laterally three complete frames (approximately 1.5 mm from midline). 
	Note that in these experiments, the imaging parameters were identical to those mentioned in the note in step 5.2.</li>
	<li>Locate the depth at which capillaries are barely visible on the view screen. Lower the plane of focus an additional 20 &#181;m to determine the top of the z-stack.</li>
	<li>Set the image thickness to 1 &#181;m. Lower the view screen for 100 &#181;m and adjust laser power throughout such that less than 1% of the pixels are oversaturated. 
	Note: the z-stack images are compiled from a series of 100 consecutive 500x500x1 &#181;m images. The larger the z-stack the more accurate the post processing calculations will be. Generally, attempt to collect a z-stack of 100 &#181;m or larger.</li>
	<li>Press the XY Repeat icon followed by the adjacent XY icon. Once the z-stack is complete, press the Series Done icon and save the file.</li>
</ol>
7. Data Processing
<ol>
	<li>Open an in vivo capillary image in the ImageJ software. Manually set the pixel to distance ratio based on values obtained from the two-photon software.
	<ol>
		<li>Select the *straight* icon from the tools menu. Draw a line extending from one capillary wall to the opposite capillary wall, and record the length provided by ImageJ. 
		Note that it may be necessary to zoom in the image in order to clearly visualize the edges of the capillary walls.</li>
		<li>Repeat step 7.1.1 at different locations along the capillary, as well as for multiple capillaries in the image.</li>
	</ol>
	</li>
	<li>Open the z-stack file into the analytical software such as Amira. Select the Filament Editor icon from the sub-application toolbar
	<ol>
		<li>Set the viewer thickness to approximately 20. Move the viewer to either the top or bottom of the z-stack.</li>
		<li>Select the Trace icon and move the cursor over the loaded image. Place nodes on the capillaries at the locations described in Figure 2C; segments will automatically appear between adjacent nodes. Trace capillaries throughout the entire z-stack. 
		Note: it will be necessary to place nodes in between end points and branch points in order to trace the capillaries throughout the z-stack.</li>
		<li>To remove these nodes, click the Remove Intermediate icon. 
		Note: This can create erroneous looped segments that must be removed manually by selecting the loop using the Select Single icon, and then selecting the Remove Selected icon. If loops continue to appear in the same area during tracing, it is likely that the nodes were placed on different capillaries.</li>
		<li>Once the tracing is complete, select the Graph Info icon to open a spreadsheet containing the automatically extracted vascular parameters. The number of nodes and segments are available on the main view screen.</li>
	</ol>
	</li>
</ol>

<ol><li>Kraft-Terry, S. D., Buch, S. J., Fox, H. S., Gendelman, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+coat+of+many+colors%3A+neuroimmune+crosstalk+in+human+immunodeficiency+virus+infection%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">A coat of many colors: neuroimmune crosstalk in human immunodeficiency virus infection.</a> Neuron. 64 (1), 133-145 (2009).</li>
<li>Ghafouri, M., Amini, S., Khalili, K., Sawaya, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-1+associated+dementia%3A+symptoms+and+causes%5BTitle%5D)+AND+%22Retrovirology%22%5BJournal%5D)">HIV-1 associated dementia: symptoms and causes.</a> Retrovirology. 3, 28(2006).</li>
<li>Antinori, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Updated+research+nosology+for+HIV-associated+neurocognitive+disorders%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Updated research nosology for HIV-associated neurocognitive disorders.</a> Neurology. 69 (18), 1789-1799 (2007).</li>
<li>Clifford, D. B., Ances, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-associated+neurocognitive+disorder%5BTitle%5D)+AND+%22The+Lancet.+Infectious+diseases%22%5BJournal%5D)">HIV-associated neurocognitive disorder.</a> The Lancet. Infectious diseases. 13 (11), 976-986 (2013).</li>
<li>Lindl, K. A., Marks, D. R., Kolson, D. L., Jordan-Sciutto, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HIV-associated+neurocognitive+disorder%3A+pathogenesis+and+therapeutic+opportunities%5BTitle%5D)+AND+%22Journal+of+neuroimmune+pharmacology+%3A+the+official+journal+of+the+Society+on+NeuroImmune+Pharmacology%22%5BJournal%5D)">HIV-associated neurocognitive disorder: pathogenesis and therapeutic opportunities.</a> Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology. 5 (3), 294-309 (2010).</li>
<li>Zimmermann, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CNS-targeted+production+of+IL-17A+induces+glial+activation%2C+microvascular+pathology+and+enhances+the+neuroinflammatory+response+to+systemic+endotoxemia%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">CNS-targeted production of IL-17A induces glial activation, microvascular pathology and enhances the neuroinflammatory response to systemic endotoxemia.</a> PloS one. 8 (2), e57307(2013).</li>
<li>Silva, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+central+nervous+system+expression+of+HIV-1+Tat+leads+to+accelerated+rarefaction+of+neocortical+capillaries+and+loss+of+red+blood+cell+velocity+heterogeneity%5BTitle%5D)+AND+%22Microcirculation%22%5BJournal%5D)">Chronic central nervous system expression of HIV-1 Tat leads to accelerated rarefaction of neocortical capillaries and loss of red blood cell velocity heterogeneity.</a> Microcirculation. 21 (7), 664-676 (2014).</li>
<li>Marker, D. F., Tremblay, M. E., Lu, S. M., Majewska, A. K., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+thin-skull+window+technique+for+chronic+two-photon+in+vivo+imaging+of+murine+microglia+in+models+of+neuroinflammation%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation.</a> Journal of visualized experiments : JoVE. (43), (2010).</li>
<li>Kleinfeld, D., Mitra, P. P., Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (26), 15741-15746 (1998).</li>
<li>Bruce-Keller, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphine+causes+rapid+increases+in+glial+activation+and+neuronal+injury+in+the+striatum+of+inducible+HIV-1+Tat+transgenic+mice%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV-1 Tat transgenic mice.</a> Glia. 56 (13), 1414-1427 (2008).</li>
<li>Zoumi, A., Yeh, A., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (17), 11014-11019 (2002).</li>
<li>Silva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transient+hypercapnia+reveals+an+underlying+cerebrovascular+pathology+in+a+murine+model+for+HIV-1+associated+neuroinflammation%3A+role+of+NO-cGMP+signaling+and+normalization+by+inhibition+of+cyclic+nucleotide+phosphodiesterase-5%5BTitle%5D)+AND+%22Journal+of+neuroinflammation%22%5BJournal%5D)">Transient hypercapnia reveals an underlying cerebrovascular pathology in a murine model for HIV-1 associated neuroinflammation: role of NO-cGMP signaling and normalization by inhibition of cyclic nucleotide phosphodiesterase-5.</a> Journal of neuroinflammation. 9, 253(2012).</li>
<li>Farber, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Region-specific+and+agent-specific+dilation+of+intracerebral+microvessels+by+volatile+anesthetics+in+rat+brain+slices%5BTitle%5D)+AND+%22Anesthesiology%22%5BJournal%5D)">Region-specific and agent-specific dilation of intracerebral microvessels by volatile anesthetics in rat brain slices.</a> Anesthesiology. 87 (5), 1191-1198 (1997).</li>
</ol>

The authors have nothing to disclose.

The method described here can be applied to analyze brain microvascular structures in a wide range of experimental models/settings. For the success of this method, three critical steps must be mastered. First, the thin-skull window must not damage the skull or underlying brain. It is easy to puncture the skull during thinning, or cause heat induced vascular leakage. This can interfere with imaging as the fluorescent dye will leak into the plane of focus and obscure the capillaries. If the skull frequently breaks during t...

Human Immunodeficiency Virus 1 (HIV-1) invades the brain during the acute phase of virus infection, and productively infects both microglia and brain resident macrophages, leading to their activation - and the release of both host-derived inflammatory mediators and soluble HIV-1 virotoxins such as Tat and gp120 (reviewed in 1,2). As a consequence, a chronic neuroinflammatory state becomes established in the CNS, which is thought to contribute to the pathogenesis of HIV-1 Associated Neurocognitive Disorders (HAND)3-5.
Chronic overexpression of HIV-1 Tat or interleukin (IL)-17A within the CNS of mice has been shown to result in microvascular rarefaction6,7. This raises the possibility that chronic neuroinflammation may contribute to the pathogenesis of HAND through effects on the cerebral vasculature. In order to further examine this question, we have developed methods to quantify cerebral vascular structures.
This paper describes a method for quantifying the number of capillary nodes, capillary segments, mean segment length, total segment length, mean capillary diameter, and total capillary volume using in vivo imaging of capillary networks through a thin skull cortical window (modified from previously described protocols)8,9, as well as ex vivo imaging of brain sections, using two-photon microscopy. This combined approach provides for a holistic quantitation of cerebral vascular parameters, since the in vivo thin-skull cortical window allows for the preservation of the cerebral environment, while ex vivo imaging of capillary networks in brain slices enables the reconstruction of complete, three-dimensional capillary networks - which can then be quantified using commercially available software.

<table><tbody><tr><td>Leica Microscope</td><td>Leica Inc.</td><td>MZ8</td><td></td></tr><tr><td>High Intensity Illuminator</td><td>Dolan-Jenner</td><td>180</td><td></td></tr><tr><td>Heating Pad</td><td>Stryker&amp;nbsp;</td><td>TP3E</td><td></td></tr><tr><td>T/PUMP</td><td>Gaymar Industries, Inc.</td><td>TP-500</td><td></td></tr><tr><td>TEC-4 Isoflurane Vaporizer</td><td>Datex Ohmeda</td><td>447</td><td></td></tr><tr><td>Artificial Tear Gel</td><td>Butler AHS</td><td>7312</td><td></td></tr><tr><td>Povidone-Iodine solution&amp;nbsp;</td><td>Aplicare</td><td>52380-1855-9</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Dumot #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td></td></tr><tr><td>Dumont #5/45 Forceps</td><td>Fine Science Tools</td><td>11251-35</td><td></td></tr><tr><td>Ferric Chloride Solution</td><td>Ricca Chemical Company</td><td>3120-16</td><td></td></tr><tr><td>Loctite 454 Prism Instant Adhesive Gel</td><td>Henkel</td><td>45404</td><td></td></tr><tr><td>Dental Cement</td><td>Stoelting</td><td>51459</td><td></td></tr><tr><td>Microtoruqe II Handpiece Kit</td><td>Pearson Dental</td><td>R14-0002</td><td></td></tr><tr><td>005 Burr for Micro Drill</td><td>Fine Science Tools</td><td>19007-05</td><td></td></tr><tr><td>Norland Blade (Dental Microblade)</td><td>Salvin Dental</td><td>6900</td><td></td></tr><tr><td>Urethane</td><td>Sigma-Aldrich</td><td>U2500</td><td>Group 2B Carcinogen</td></tr><tr><td>Braided Suture</td><td>Ethicon</td><td>735G</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>&amp;nbsp;Arterial Catheter</td><td>SAI Infusion Technologies</td><td>MAC-01</td><td>The end of the catheter was manually stretched out in order to decrease its diameter.&amp;nbsp;</td></tr><tr><td>Blood Pressure Moniter</td><td>World Precision Intruments</td><td>SYS-BP1</td><td></td></tr><tr><td>Blood Pressure Transducer and Cable</td><td>World Precision Intruments</td><td>BLPR2</td><td></td></tr><tr><td>RAPIDLab Blood Gas Analyzer&amp;nbsp;</td><td>Siemens&amp;nbsp;</td><td>248</td><td></td></tr><tr><td>40 &amp;mu;l Capillary Tube</td><td>VWR</td><td>15401-413</td><td></td></tr><tr><td>Texas Red-dextran (70,000 MW, 10 mg/kg dissolved in saline)</td><td>Invitrogen</td><td>D-1830</td><td></td></tr><tr><td>Adult Mouse Brain Slicer Matrix</td><td>Zivic Instruments</td><td>BSMAS001-1</td><td></td></tr><tr><td>Olympus Fluoview 1000 AOM-MPM Multiphoton Microscope</td><td>Olypmus</td><td>FV-1000 MPE</td><td></td></tr><tr><td>MaiTai HP DeepSee Ti:Sa laser</td><td>Spectra-Physics</td><td></td><td></td></tr><tr><td>ImageJ Software</td><td>National Institutes of Health (NIH)</td><td></td><td>Available at http://rsb.info.nih.gov/ij/download.html</td></tr><tr><td>Amira Software</td><td>Visage Imaging&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

quantification of cerebral vascular architecture using two-photon microscopy in a mouse model of hiv-induced neuroinflammation

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

The thin-skull cortical window allows for in vivo two-photon imaging of cortical capillaries (Figure 1). A suitable area to image shows numerous, distinct capillaries (Figure 1A). In the same field of view, there is no arterial cell wall autofluorescence, and there may be other fluorescent signals, such as collagen fluorescence, induced by second harmonic generation11 (Figure 1B).
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This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

The overall goal of this procedure is to quantify changes in the cortical capillary networks after prolonged exposure to the HIV virotoxin, tat. This method can help to examine key changes in cortical capillary morphology which may contribute to the pathogenesis of HIV associated neurocognitive disorders. The main advantage of this technique is that it allows for physiologically accurate quantification of multiple capillary morphological parameters.Though this method can provide insight into the pathogenesis of HIV associated neurocognitive disorders. It can also be applied to other diseases in which vascular changes occur such as Alzheimer's disease. Begin this procedure by applying artificial tear gel to the eyes of an anesthetized mouse.Next, shave its head using an electric razor. Then, apply povidone iodine solution to sterilize the scalp, and allow it to dry. Under a light microscope, remove the scalp of the animal, to completely expose the parietal bones, the caudal frontal bones, and the bregma point.Apply a small quantity of a ten percent ferric chloride solution to the skull, in order to dry the membrane for easy removal. Subsequently, remove the dried membrane, by gently scraping the skull with the forceps. Then, apply a thin layer of glue around the head plate window.Gently press the head plate against the skull of the mouse, to keep the area of interest in the center of the window. Afterward, apply a drop of dental cement to the head plate, to polymerize the glue. Next, apply a thin layer of glue along the edge of the head plate window to create a reservoir to hold the saline.Once the glue has dried, screw the head plate into the mouse head plate harness. Remove any glue from the head plate window using a drill bit attached to a micro-torp drill, set at 6, 000 rpm. Stop every 10 to 15 seconds to prevent overheating of the mouse cranium.After that, using a new drill bit, begin to thin the skull using a micro-torp drill set at 4, 000 rpm. Move the drill gently across the skull without direct downward pressure. Once the skull is completely thinned, detach the mouse from the holder and place it on its back.Gently tape down both hind limbs to clearly expose the medial thighs. Then remove the hair over both medial thighs and disinfect the surgical site by covering the thighs with povidone iodine. Next, gently remove the skin on the medial right thigh, above the femoral vein and artery.Apply approximately three to five drops of 0.9%saline to the surgical site. Then, separate the femoral vein from the artery, by bluntly dissecting into femoral neurovascular bundle. Now, place two three centimeter pieces of surgical suture, underneath the femoral artery, approximately one centimeter apart.Twist the upper suture clockwise to create a vascular tourniquet that will help to prevent excessive blood loss during catheterization. Subsequently, make a small incision in the femoral artery using spring scissors, where the catheter will later be inserted to monitor the mouse's physiological parameters. In this procedure, remove the skin on the medial left thigh of the mouse.Next, locate the femoral vein. Using a one milliliter syringe and a 30 gauge needle, draw up 130 microliters of the fluorescent dye solution. Inject 100 microliters of the dye slowly into the femoral vein.After removing the needle, apply steady gentle pressure to the injection site to stop any bleeding and allow the dye to circulate for five minutes. Then, close the surgical site with sutures. Carefully flip the mouse onto its stomach and place it into the mouse head plate harness.For in vivo two photon imaging, move the surgical apparatus to the two photon microscope and make sure to maintain the animal's anesthesia level. Next, place a small amount of 0.9%saline into the head plate reservoir and lower microscope objective so that it comes into contact with the saline. Then, locate the area of interest using the bright field viewing objective.Afterward, begin two photon imaging. Locate a capillary bed on the view screen and magnify this area using the optical zoom, too. Acquire images of the capillaries using the two photon imaging software.For ex vivo two photon imaging, make an incision in the scalp from the interparietal bones to the frontal bones of the decapitated mouse. Secure the skin to the sides of the skull with the index finger and the thumb, place the extra fine scissors underneath the medial interparietal bone and cut the skull along the sagittal suture until approximately three millimeters after the bregma point. Then, separate the skull from the brain and carefully remove any meningees from the surface of the brain with the forceps.Gently slide the forceps under the brain to free it from the skull. Afterward, place the brain in a brain matrix, specific for mice, and wash it with drops of ACSF. Next, remove a millimeter thick coronal brain section.Place the brain section onto a concave glass slide containing ACSF, with the most anterior part of the section facing upwards. Then gently cover the brain slice with a glass cover slip. Transfer the slide to the microscope stage and place a small quantity of 0.9%saline on the cover slip.Lower the microscope objective until it comes in contact with the saline. Subsequently, locate the mid-line of the brain using the bright field objective. Now, begin two photon imaging, and locate the mid-line again using the 25x objective.Accomplish this by looking for the longitudinal fissure at the cortical surface of the coronal section. Then, place the right edge of the imaging screen on the mid-line and move the viewer screen laterally over three complete frames. Locate the depth at which capillaries are barely visible on the view screen.Then, lower the plane of focus for an additional 20 micrometers to determine the top of the z-stack. Set the image thickness to one micrometer. Lower the view screen for 100 micrometers and adjust the laser power throughout such that less than one percent of the pixels are over-saturated.Finally, acquire the z-stack. In this figure, after the brain slice has been prepared, an imaging area at approximately 1.5 millimeters from the mid-line is located. A 100 micrometer z-stack produces a three dimensional image used for analysis in the Amira Analytical Software.The capillary network is then manually traced by placing nodes at the beginning and end of a capillary or any location where one capillary branches into another. This produces a fully skeletonized image from which the morphological parameters are automatically extracted. The number of capillary nodes, segments, mean segment length, and the total segment length can be extracted using two photon ex vivo imaging of mouse brain slices.Here, a two dimensional image of the capillary networks is produced by in vivo imaging, in which the capillary diameter can be extracted and the total capillary volume can be calculated using the capillary diameter and the total segment length obtained from the ex vivo imaging. Once mastered, this procedure can be completed in three and a half hours if it is performed properly. While performing this procedure, it's important to move quickly while maintaining a steady level of anesthesia as isoflurane can cause vasodilation of cortical capillaries, thus skewing the data.During this procedure other capillary parameters may be obtained, such as red blood cell velocity or red blood cell flux, which can provide a deeper understanding of the pathogenic changes seen in HIV associated neurocognitive disorders and other neurodegenerative diseases. Good luck with your experiments.

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Research

JoVE Journal

Neuroscience

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Learn two-photon microscopy to quantify cerebral vascular architecture in a mouse model of HIV-induced neuroinflammation. This method enables accurate assessment of cortical capillary changes.

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

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