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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+coat+of+many+colors:+neuroimmune+crosstalk+in+human+immunodeficiency+virus+infection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+associated+dementia:+symptoms+and+causes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Updated+research+nosology+for+HIV-associated+neurocognitive+disorders.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-associated+neurocognitive+disorder.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-associated+neurocognitive+disorder:+pathogenesis+and+therapeutic+opportunities.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-targeted+production+of+IL-17A+induces+glial+activation,+microvascular+pathology+and+enhances+the+neuroinflammatory+response+to+systemic+endotoxemia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin-skull+window+technique+for+chronic+two-photon+in+vivo+imaging+of+murine+microglia+in+models+of+neuroinflammation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+and+stimulus-induced+changes+in+blood+flow+observed+in+individual+capillaries+in+layers+2+through+4+of+rat+neocortex.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphine+causes+rapid+increases+in+glial+activation+and+neuronal+injury+in+the+striatum+of+inducible+HIV-1+Tat+transgenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+and+extracellular+matrix+in+vivo+by+using+second-harmonic+generation+and+two-photon+excited+fluorescence.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+and+agent-specific+dilation+of+intracerebral+microvessels+by+volatile+anesthetics+in+rat+brain+slices.">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 the thin-skull preparation, it is most likely caused by too great a downward pressure. Holding the microtorque drill as close as possible to the burr allows for greater tactile control that will decrease the downward pressure on the skull.
Second, arterial catheterization should occur as quickly as possible once the artery has been cut. Failure to insert the catheter quickly can cause excessive blood loss that will compromise the physiological integrity of the mouse. Difficulty inserting the catheter is usually due to the relatively large size of the catheter compared to the femoral artery. It may be necessary to stretch the catheter in order to reduce its diameter. Additionally, the tips of the #5 forceps can be used to grab and enlarge the opening made in the artery to ease in the placement of the catheter.
Third, the duration of the in vivo surgical procedures should be minimized so that the urethane anesthesia can be administered as soon as possible. The isoflurane anesthesia can cause vasodilation of cerebral blood vessels13, thus skewing the capillary diameter data.
It should be acknowledged that the Amira analytical software can automatically extract the radii of manually traced vessels; however, when using this feature to analyze z-stack images from ex vivo brains, the value is inaccurate. This is because, after removing the brain, the cerebral capillaries are no longer pressurized, and may become morphologically distorted. The in vivo capillary diameter measurement circumvents this problem because the capillaries are pressurized under normal, physiological conditions.
It must also be noted that this method is not without limitations. First, significant training is required to master the in vivo imaging preparation. A researcher must learn to efficiently perform two technically challenging techniques (thin skull cortical window and arterial catheterization); an error in either technique can compromise the experiment and invalidate the data. Second, analysis of the ex vivo z-stacks is time-intensive. The skeletonization of one z-stack image can take up to 2.5 hr. Furthermore, it is advisable that this analysis be completed by an experienced researcher to ensure consistency of the skeletonization process (which involves careful manual tracing of blood vessels).
Once all aspects of this protocol are mastered, the data obtained provide a more in-depth and physiologically accurate quantitation of vascular parameters in comparison to other traditionally used methods that show capillary density as capillaries per unit volume, or capillaries per field of view. The in vivo imaging technique allows for the study of other parameters including, but not limited to, red blood cell velocity, arteriole dilation, and red blood cell flux7,12, which may also be able to be examined in longitudinal studies. Furthermore, it can easily be adapted and modified to study research questions related to brain microvascular structure. Such studies could include quantitating pathogenic changes in capillary morphology in other neurocognitive diseases, or measuring age-related changes in capillary density. Therefore, the method presented in this paper is a versatile and powerful tool for the quantitative analysis of cerebral vascular architecture.

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

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&#160;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).
Once the preparation of the brain slice is complete, an imaging area approximately 1.5mm from midline is located (Figure 2A). A 100 &#181;m z-stack produces a three-dimensional image used for analysis in the Amira analytical software (Figure 2B). 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 (Figure 2C). This produces a fully skeletonized image from which morphological parameters are automatically extracted (Figure 2D). The number of capillary nodes, segments, mean segment length, total segment length, and total capillary volume can be extracted using two-photon ex vivo imaging of mouse brain slices (Figure 2).
Figure 3 shows representative results obtained using the method presented in this paper. In this experiment, a transgenic mouse model of HIV neuroinflammation was used10. Production of the HIV virotoxin Tat was induced in Tat+ mice by doxycycline-infused food for 12 weeks. The control group consisted of Tat- littermates fed the same food. There was no statistically significant difference between Tat+ and Tat- mice in any of the capillary parameters measured. Thus, 12 weeks&#39; exposure of brain tissue to HIV-1 Tat is insufficient to cause rarefaction of the brain microvasculature. In contrast, our previously published data show that more prolonged (20 weeks) chronic CNS expression of Tat results in rarefaction of cerebral vasculature7. Thus, the data shown here (Figure 3) provides valuable new information in regards to the kinetics of Tat-induced vascular remodeling within the CNS of mice.
<img alt="Figure 1" src="/files/ftp_upload/53582/53582fig1.jpg" /> 
Figure 1. Acquisition of cortical capillary diameter. (A) Following a thin-skull cortical window preparation and intravenous fluorescent dye injection, two-photon microscopy was used to image cerebral vasculature. In our experiments, we used a dextran conjugated to a red emitting rhodamine dye that was excited at 780 nm, and fluorescence was visualized through a 607/36 bandpass emission filter. (B) The same field of view should show no arterial autofluorescence as detected through a 480/20 bandpass emission filter. Second harmonic generation may induce other fluorescent signals to appear through the filters11. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53582/53582fig2.jpg" /> 
Figure 2. Generation of skeletonized capillary networks.&#160; (A) Mice were intravenously injected with a fluorescent dye and sacrificed. A 2 mm coronal section of brain was taken between bregma 0 and bregma -2, and imaging was performed at the 0 bregma approximately 1.5 mm from midline. A representative image of the cortical location chosen for imaging (black box) from a C57BL/6 mouse is shown. This region, the somatosensory cortex, was chosen because we have previously shown that dysregulation of cerebral blood can occur at this site in Tat+ mice12. (B) Representational three dimensional z-stack images of capillary networks. (C) Diagram of the method used to manually trace capillaries during vessel quantification. (D) Representative image of skeletonized z-stack image. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53582/53582fig3.jpg" /> 
Figure 3. Quantification of cerebral vascular architecture in Tat- mice versus mice exposed to the candidate HIV virotoxin Tat for 12 weeks.&#160; Mice with a doxycycline (DOX) inducible HIV-1 Tat transgene driven by the astrocyte-specific glial fibrillary protein (GFAP) promoter (HIV Tat-Tg mice) were used to examine the effect of HIV-1 induced neuroinflammation on cerebral vascular structure, as described7. These mice (Tat+, n=3) were exposed to a chronic (12 week) regimen of Tat induction (i.e., 12 weeks of DOX). Tat- mice (n=4) were used as age-matched controls (these mice correspond to non-transgenic littermates of the Tat+ mice, and therefore do not express HIV-1 Tat). Like the Tat+ mice, the Tat- mice also received 12 weeks of DOX exposure. In the mice exposed to Tat for 12 weeks (Tat+), versus those not exposed to Tat (Tat-), there was no statistically significant difference in the number of nodes (A), the number of segments (B), mean segment length (C), or total capillary length (D), capillary diameter (for the Tat+ mice, we analyzed 14 capillaries in 6 imaging areas; for Tat- mice, we analyzed 7 capillaries in 4 imaging areas) (E), or total capillary volume (F). Since the data were not normally distributed (as determined by the Shapiro-Wilk test), the exact Wilcoxon rank sum test was used to calculate statistical significance (determined in this case as P&#60;0.05). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53582/53582fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation at JoVE.com

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

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 ...

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9.2K Views.  University of Rochester Medical Center. 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.

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

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or thinned-skull 3 preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX3CR1 GFP/+ mice 4 to visualize microglia with enhanced green fluorescent protein to &le;150 &mu;m beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull1,2 or ...

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Medicine

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

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

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

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

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

In Vivo Three-Dimensional Two-Photon Microscopy to Study Conducted Vascular Responses by Local ATP Ejection Using a Glass Micro-Pipette

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the regulation of cerebral blood flow (CBF) is incompletely understood, especially at the capillary level. Two-photon microscopy is a powerful tool widely used to study CBF and its regulation. Currently, this field is limited by the lack of in vivo two-photon microscopy studies examining (1) CBF responses in three-dimensions, (2) conducted vascular responses, and (3) localized interventions within the vascular network. Here, we describe a 3D in vivo method using two-photon microscopy to study conducted vascular responses elicited by&#160;local ejection of ATP with a glass micro-pipette. Our method uses fast and repetitive hyperstack two-photon imaging providing precise diameter measurements by maximal intensity projection of the obtained images. Furthermore, we show that this method can also be used to study 3D astrocytic calcium responses. We also discuss the advantages and limitations of glass micro-pipette insertion and two-photon hyperstack imaging.

We present an optimized local ejection procedure using a glass micro-pipette and a fast two-photon hyperstack imaging method, which allows precise measurement of capillary diameter changes and investigation of its regulation in three dimensions.

Maintenance of normal brain function requires a sufficient and efficient supply of oxygen and nutrition by a complex network of vessels. However, the ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

Immunology and Infection

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

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

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A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

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High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

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

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In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus