In adult patients infected with HIV neuroinflammation interferes with normal synaptic signaling leading to neurocognitive deficits that include difficulties with abstraction, verbal fluency, attention learning, new information and memory. HIV one tat induces the secretion of inflammatory molecules, which can mimic the synaptic dysfunction seen in patients with HIV here. Adult mice expressing GFP in mononuclear cells, including microglia, are used to investigate HIV one tat induced neuroinflammation using thin skull two photon microscopy.
The responses of microglia following injection of tat can be imaged in vivo. The resulting images demonstrate shortening and thickening of microglial processes and the formation of phagocytic cup like structures as well as peripheral monocyte trafficking in cerebral micro vessels. Hi, I'm Dan Marker from the lab of Harris Galbard and the Center for Neural Development and Disease at the University of Rochester.
Hi, I'm EF Trombley from the Laboratory of Esca in the Department of Neurobiology and Anatomy at the University of Rochester. Hi, I'm Shain Luu, also from Albas Lab. Today we'll show you a procedure for two photon imaging using a thin skull preparation.
We use this procedure to study chronic neuroinflammation. So let's get started In the experiment shown here. OT express GFP in mononuclear cell lineages in including microglia are used.
Different strains of mice can be used to meet the needs of a specific project. Begin preparing a fentanyl, midazolam meine anesthetized mouse for surgery. First, ensure that the animal no longer responds to painful stimuli such as a tail pinch.
Place the animal on a heating pad to prevent post anesthesia hypothermia. Then cover the animal's eyes with a protective ophthalmic ointment to keep the eyes moist. All surgical instruments should have been autoclave or sterilized with a glass bead sterilizer further disinfect the instruments by soaking them in 70%ethanol.
Using scissors, remove the hair from the animal's scalp, then disinfect the area using a 10%povidone iodine solution and 70%ethanol. Begin the surgery using small scissors to make a midline incision in the scalp, starting four to five millimeters, cordal to the skull and advancing forward to the front of the eyes. This incision should be long enough that the skin will not interfere with the gluing of the plate that stabilizes the mouse head during two photon imaging.
Next, apply 100%ethanol the sterile cotton swab to dry the membranes on top of the skull. Scrape them away using fine tweezers. If the membranes are not removed, the head plate attachment will be unstable.
Under a dissecting scope, identify the area to be thinned. Avoid areas of interest directly located over cranial sutures as the skull is less stable in these areas and underlying large vessels and meninges will interfere with imaging sent to the viewing window on the area of interest. Then again, using a cotton swab, try the skull again using a 10%solution of ferric chloride.
Place a thin layer of the perma bond, nine 10 glue around the edges of the viewing window underneath the head plate. Then place the plate over the area of interest on the animal's skull with light pressure to bond the glue. Apply a small amount of acrylic around the edge of the viewing window using a syringe.
Finally, apply a small amount of Tite 4 54 to the edges of the viewing window. To prevent leaks. Screw the head plate to the animal holder and check the stability under a dissection microscope By lightly probing with a pair of forceps, there should be no movement of the skull relative to the head plate.
Place a drop of saline on the viewing window to ensure that there are no leaks in preparation for thinning. Dry the skull using a combination of sterile cotton swabs and compressed air. Thin the skull using an IRF 0 0 7 drill bit in a micro to two drill.
At 4, 000 RPM. Very gently begin thinning a two to 2.5 millimeter diameter circular area. Use only light sweeping motions nearly parallel to the skull.
Do not use direct downward pressure. Stop drilling every 20 to 30 seconds to remove bone dust using the compressed air. These breaks in drilling allow the skull to cool so that there is no heat induced damage to the underlying brain tissue.
As the drilling progresses, notice the transition to the moister spiny bone layer called the Diplo. Once this layer is reached, exercise extra caution with the drill. Occasionally check the thinness of the skull by placing saline over the thin area and viewing under the dissecting scope.
Saline will further enable heat dissipation. As the skull is thinned, smaller vasculature becomes visible. Use a dental microblade to achieve the final thinning under saline.
The microblade provides much more tactile feedback about the stability of the skull. This allows for much thinner preparations than with the drill alone. The optimal skull thickness for imaging is 10 to 30 micrometers.
The entire thinning process normally takes between 15 and 30 minutes. Check the thinness of the skull under epi fluorescence multiple times while making a thin skull window. The sharpness and depth of visible microglia and vasculature give a good indication as to when the window is ready for imaging.
Once the window is ready, use a one millimeter diameter glass pipette to place a small drop of Cy acrylic glue onto the thin skull area and carefully lower a piece of cover glass onto it. Then press gently against the skull to squeeze out any excess glue, avoiding bubble formation between it and the cover glass, which may obstruct the view. Once dry, use a microblade to carefully remove any glue left on top of the cover glass in preparation for two photon imaging.
Cover the thin skull cortical window with saline and locate the area of interest under epi fluorescence. To enable subsequent imaging of the area, take a picture using a photographic camera for imaging. Shown here a custom made two photo microscope with a ti sapphire laser tuned to 920 nanometers is used.
Fluorescence is detected by using a photo multiplier tube or PMT in whole field detection mode and a 5 81 80 emission filter. A 20 x water immersion lens is used throughout the imaging session. Set the maximum output of the laser power of the objective between 50 and 65 milliwatts.
Next, select an area of interest either cortical layer one or two up to 150 micrometers below the peel surface. To facilitate re-imaging, take pictures of the same field of view at one x at two x and three x digital zoom. Blood vessels can servee as landmarks to easily find the same field of view during subsequent imaging sessions.
Under a zoom of three x, acquire multiple Zacks with 80 to 90 Z steps each and a 0.69 micrometer step every five minutes for up to 30 minutes. This enables a good sampling of microglial morphology and behavior over time between acquisitions of Zacks, verify the level of saline and the depth of anesthesia. If necessary, a booster dose of one third of the original dose of anesthetic cocktail can be given during the procedure to prevent tat deposition onto the syringe and needle during the injection days before injection.
Siloized the inner lining of a 35 gauge needle and 10 microliter micro volume syringe by withdrawing sizing solution until it fills both the needle and syringe. Allow the solution to sit for five minutes at room temperature, then empty the solution from the syringe, remove the plunger and allow the syringe and needle to completely dry. Finally, wash the syringe and needle thoroughly with deionized water.
Once siloized, a syringe and needle can be used for more than six months before a recoding is necessary. On the day of injection, place a small drop of the TAT solution on a siloized glass slide. Using a siliconized pipette tip, load the desired amount of solution into the micro volume syringe from this drop.
Using a drill bit, perform a small craniotomy 0.5 millimeters in diameter, either three millimeter rostral or lateral to the thin skull cortical window where two photon images have been obtained. To accomplish this, carefully thin the skull. Then use a sharp pair of forceps to remove the thin layer of bone.
Use a micro manipulator to line up the sized 35 gauge needle and syringe with the craniotomy. Then carefully advance the needle to a depth of 700 to 900 micrometers below the peel surface. Deliver three microliters at 80 nanoliters per minute using a microprocessor controlled syringe pump.
This figure shows GFP labeled microglia visualized with two photon imaging after implantation of the thin skull cortical window, approximately 50 micrometers below the peel surface. The single two photon sections taken on day zero, 15 to 30 minutes after implantation of the thin skull cortical window, as well as those taken seven and 17 days later demonstrate that the windows stay clear while microglia remaining activated in the absence of inflammatory insults. This highlights the utility of this method for investigating both normal and inflammatory interactions between immune effector cells and other neural cell types.
In vivo the Z projections taken at six and 24 hours post-injection of the HIV neurotoxin TAT exhibit marked morphologic changes of activated microglia. Further demonstrating our ability to study morphologic changes of microglia in real time in acute and chronic models of neuroinflammation. We've just shown you how to prepare a thin skull window and use it to study neuroinflammation When doing this procedure.
It's important to remember to take your time when tending the skull to prevent any damage caused by the preparation itself. So that's it. Thanks for watching and good luck with your experiments.
