The brain vasculature is a complex network of penetrating arterials, capillaries, and ascending venules. These vessels are encased by mural cells, such as ensheathing pericytes, which express alpha smooth muscle actin, and are found within the first four branches from penetrating arterials. We use Acta2-RCaMP 1.07 transgenic mice, to visualize calcium transients in these cells, through the expression of the genetically encoded calcium indicator RCaMP.
Calcium plays a strong role in contraction of these cells and blood flow control. Capillary pericytes are found farther downstream in the vascular network and can be labeled in other transgenic models. However, we focus on ensheathing pericytes in this method.
Our mice have been surgically implanted with a chronic cranial window that permits access to the brain vasculature for imaging. Here, we outline the necessary preparation steps and tail vein injection followed by the two photon imaging and analytical methods needed to acquire pericyte calcium data and information about blood flow in anesthetized animals. The following items are required for a tail vein catheter injection:insulin syringes, a 15 centimeter piece of PE10 tubing, 30 gauge needles, gauze, saline, forceps, green fluorescein dextran dye, pliers, and scissors.
We also have a needle with ketamine Xylazine anesthesia that we will inject before imaging as it is better than isoflurane for blood flow measurements. Using the pliers, break a 30 gauge needle from the hub. Carefully insert the needle into the end of the piece of PE10 tubing attached to a saline filled syringe.
This is the catheter for injection. Anesthetize a mouse with isoflurane and apply I-Lube gel. Place a glove field with warm water on the tail to dilate the lateral vein.
After cleaning the tail with ethanol, insert the needle into the vein and gently inject saline through the catheter to ensure the needle is placed correctly. Switch the syringe on the catheter for a syringe filled with fluorescein dextran dye. Slowly inject the dye to make sure no bubbles enter the tube.
Replace the dextran syringe with the saline syringe and rinse the tubing until no dye is left in the tube. Remove the needle and press with gauze. Inject ketamine Xylazine anesthesia for imaging.
With the mouse head fixed on a heating pad, clean the cranial window with dental applicators. Apply ultrasound gel on the window and focus through the two photon microscope objective. For two photon imaging, we use a microscope with tuneable titanium Sapphire laser for fluorescents, excitation, and photomultiplier tubes for emission detection.
In the microscope software, set the wavelength to 990 nanometers to excite both RCaMP and fluorescein dextran. Next set the laser power by adjusting the pockel cell voltage and the PMT detector sensitivity. Live scanning with these parameters and higher resolution, RCaMP positive mural cells and the fluorescently labeled blood plasma can be seen.
Acquisition of a depth stack is recommended to properly locate pericytes in the vascular network. When focused at the top of the tissue near the PO vessels, set this as the zero point and top of the Zed series stack, then focus down in the tissue to the desired depth and set this as the bottom of the stack. The laser power must be adjusted to increase as the microscope moves deeper through the stack.
Opening the Zed series in image processing software, merge the two channels as colored images and scan through the stack for pericytes and blood vessels of interest. Select regions of interest that contain pericytes and save the positions to help with locating these spots again in future imaging sessions. To collect and move a pericyte calcium event, increase the acquisition frame rate to more than 10 frames per second.
Set the imaging duration to 60 seconds and update the save path with a unique file name. Zoom optically on the vessel to account for the lower resolution and to gain a closer view of the pericyte. Acquire the T series.
To measure blood vessel diameter and red blood cell velocity, select line scan to start a one dimensional scan with the microscope. First set the duration of the scan in milliseconds. Then draw a line that bisects the vessel of interest and moves parallel along the vessel.
This will generate a kymograph of the vessel diameter on the left and streaks of the red blood cells moving through the vessel on the right. See the materials table for a complete list of programs and packages used in this protocol. First select regions of interest by hand in image processing software.
Load the T series file, take the average of the stack, and make a colored image. Select the polygon tool and outline the visible ensheathing pericyte structures such as soma and processes. Give each region of interest a unique name and save them as a zip folder that can be loaded later in the programming software.
Open the programming software and make sure the folders for the image processing packages are on the path. Import the calcium T series into the programming software and define what is on each channel. In this case, it is a cellular signal on channel one and blood plasma on channel two.
Plot the data as a movie within the programming software to facilitate visualization. For removing green fluorescents from fluorescein dextran that bleeds through into the red RCaMP channel, un-mix the channels in the image processing package. First select a region that only contains fluorophore 1, RCaMP in this case.
Second select a region that only contains fluorophore 2, fluorescein in the plasma. Finally select a background area that does not have either fluorophore. This generates a spectral contribution matrix, which is applied to each pixel in each channel.
It significantly improves the localization of the RCaMP signal, which will enhance the detection of calcium events in these structures. There are multiple ways that the calcium imaging data can be analyzed within the image processing packages. First run the cellular signaling analysis on the unmixed calcium movie and load the regions of interest from the zip folder that were selected from the pericytes by hand earlier.
Set the scale factor to one because the regions do not need to be resized. After processing, generate plots of each ROY and the normalized calcium traces in different colors. If the code does not detect the majority of calcium events in the individual traces, modify the built-in parameters within the optimization box, such as decreasing the threshold for the short pass filtered data to three times the standard deviation of the baseline period, which is the first 30 frames of the T series.
In order to detect and classify the signals, the normalized calcium trace is long pass and band pass filtered, which helps to smooth the data for amplitude and with estimations, but also to determine if signals are single peaks, multi peaks or plateaus. Output the data as a CSV file for further statistical analysis. Run the cellular signaling analysis a second time on the unmixed calcium movie and select the automated region of interest identification based on the activity and change in fluorescents in three dimensions, X, Y, and time.
Plot the process results to view the identified regions of interest is different colors. If the algorithm does not detect ROYs that are clearly visible by eye in the movie, modify the built-in parameters, such as increasing the Gaussian filter that smooth the data in time and decreasing the threshold for finding ROYs to three times the standard deviation of the baseline. Plot the ROYs as a movie for further visualization.
Output the data as a CSV file for further statistical analysis. Import the line scan kymograph data file into the programming software and define what is on each channel. Run the diameter analysis function on the line scan, which opens a box to select the area that corresponds to the diameter in the kymograph.
Draw a box outside the kymograph fluorescents'boundaries, that corresponds to the vessel diameter. Process this data class in order to measure the full width at half maxima for vessel diameter and generate a plot. Next run the velocity rate on transformation analysis and draw a box inside the border of the kymograph fluorescents.
Process this data class in order to calculate the velocity, flux, and linear density of red blood cells. Output the blood flow results as a CSV file for further analysis. Selecting cellular structures by hand permits detection of calcium fluctuations within these regions, including different types of signal peaks, such as single peaks and multi peaks, after the normalized calcium traces are low pass and band pass filtered.
Additionally, regions of interest are identified by grouping active pixels together where the fluorescents'intensity changes over time using image processing algorithms. This can be applied to any dynamic cellular signal by adjusting the time, threshold, and spatial parameters to encompass the expected size and shape of the signal. Decreasing the threshold for signal identification finds more regions of interest.
Clear, hemodynamic kymographs are analyzed to measure diameter and red blood cell velocity in blood vessels near ensheathing pericytes. The diameter is calculated from the full width at half maximum of the fluorescents. The red blood cell velocity is approximated from the streaks made from unlabeled red blood cells, where the angle is input into a radon transformation to calculate the velocity.
Poor quality kymographs, where there is fluorescent saturation, poor signal to noise ratio, or movement of the imaging field, creates unreliable plots with error points, indicated as red crosses, where data can not be determined. The quality of the acquired data is critical for a good outcome and following the steps described in this protocol ensures good results. In this video, we outline the procedure for combined calcium imaging of brain ensheathing pericytes and blood flow measurements by two photon microscopy.
These techniques are useful for addressing questions about mural cell physiology and localization within the brain vascular network, but they can be adapted to study calcium transients in any cell type in the brain or other organ system.
