This method will provide better insight into the function of parenchymal arteriolar endothelium which directly links intracerebral blood flow to the fueling of neuronal and glial activity throughout the brain. A distinct technical advantage over arteriole endothelium is the enhanced resolution of morphological dimensions and calcium and electrical signaling among individual intracerebral endothelial cells and their respective organelles. Initially, isolation and examination of intracerebral endothelium will be very challenging.
However, it is possible to master this technique with plenty of practice accompanied by patience and dedication. Wash the isolated brain with a cold dissection solution in a beaker or Petri dish to remove remaining blood from the surface of the brain. Place the ventral brain side facing up in a chamber containing cold dissection solution to isolate parenchymal arterioles.
To isolate parenchymal arterioles, secure the isolated brain with steel pins in cold dissection solution in a Petri dish containing more than 50 centimeter deep charcoal-infused silicon polymer coating. Cut a rectangle of brain tissue from both hemispheres with sharp and aligned dissection scissors around the MCA while ensuring that the upper part of the tissue segment is past the branching point from the circle of Willis. Secure the brain tissue into the dish with the MCA facing upwards with steel pins.
Carefully make a shallow incision near the pins and remove the pia with small forceps, gently peeling from one end towards the other. Carefully secure the isolated pia with parenchymal arterioles branched from MCA in the dish with the pins and carefully dissect the parenchymal arterioles. Cut off any remaining distal branches and ensure the arteriole is clean with no tissue attached.
Use this clean intact arteriole for enzymatic digestion. Alternatively, cut each arteriole into two pieces for enzymatic digestion to prepare endothelial tubes if desired. For partial digestion of arteriolar segments, place intact arteriolar segments into one milliliter of dissociation solution in a 10 milliliter glass tube containing papain, dithioerythritol, collagenase, and elastase.
Incubate arteriolar segments at 34 degrees Celsius for 10 to 12 minutes. To isolate the arteriolar endothelial tube, place the trituration pipette attached with the micro syringe injector in the dissociation solution in the chamber and position it close to one end of the digested vessel segment. Set a rate within the range of one to three nanoliters per second on the pump controller for gentle trituration.
While maintaining 100X to 200X magnification, withdraw the arteriolar segment into the pipette and then inject to dissociate the adventitia and smooth muscle cells. Triturate the vessel segment until all smooth muscle cells are dissociated and only endothelial cells remain as an intact tube. With micro manipulators, secure each end of the endothelial tube on the glass coverslip in the chamber with borosilicate glass pinning pipettes To measure endothelial membrane potential, while viewing through the 4X objective, carefully position the sharp electrode tip just over a cell of the arteriolar endothelial tube into the flowing physiological salt solution with a micro manipulator.
Gradually increase magnification to 400X and reposition the electrode tip as needed. Using the micro manipulator, gently insert the tip of a sharp electrode into one of the cells of the endothelial tube and start recording VM using an electrometer. Once the endothelial resting VM is stable from minus 30 to minus 40 millivolts, apply the desired pharmacological agents per experimental objective.
Load the endothelial tube with the fluorescence tracker for the plasma membrane or desired organelle at 37 degrees Celsius for 15 to 30 minutes. Wash the cells with fresh superfusion physiological salt solution and image live cells under the microscope at the excitation wavelength of the respective dyes. Endothelial function was assessed by measuring intracellular calcium and membrane potential in response to a pharmacological agent MTA, a potent purinergic receptor agonist at 37 degrees Celsius.
Upon application of one micromolar MTA, intracellular calcium concentration rapidly increases with a concomitant hyperpolarization. Furthermore, the intact endothelium was incubated at 37 degrees Celsius with fluorescent trackers for visualization to examine cellular morphology. Live endothelial cell imaging showed co-staining for plasma membrane and nuclei.
The plasma membrane was stained together with an endoplasmic reticulum fluorescent stain whereby areas of apparent overlap of ER within proximity of the plasma membrane appear orange. The experimenter should be careful during dissection and isolation to avoid damage to the arterioles because a damaged arteriole will definitely not yield an intact endothelial tube. Following the procedure, the cells may be used for fixed immunohistochemistry with fluorescent antibodies or live cell staining of organelles and membrane lipids.
This technique will generate new information for understanding mechanisms underlying cerebral blood flow at the fundamental level for physiology and select transitions towards pathology for therapy.
