The overall goal of this procedure is to efficiently and expeditiously dissect the peel and arachnoid portions of the meninges and the associated arterial vasculature, overlaying the surface of the forebrain cortex and midbrain, thalamus, and calli, as well as to harvest the choroid plexus from the lateral ventricles. This is accomplished by first removing the pineal gland and separating the meninges and arterial trees coddle to the forebrain. Second, the arteries and arterials on the cortical surface are carefully removed, starting at the ventral circle of Willis, and then proceeding to the dorsal cortical surfaces so that the peel and remaining arachnoid membranes stay attached to the vasculature, making sure to minimize the harvesting of adjacent cortical surface tissues.
Next, the cortex and commissural fibers are dissected away from the septum and hippocampus, exposing the lateral ventricles to allow the harvesting of the choroid plexus. Finally, the hippocampus is removed, exposing the thalamus and calli of the midbrain. The meninges and associated vasculature overlying this region are then dissected.
Ultimately, the integrity and specificity of the dissected meninges and the associated arterial vasculature, as well as the choroid plexus, can be determined by comparing the gene expression profiles of each of these regions. This method can help answer key questions in research related to the vascular that supports brain function. It includes similarities and comparisons and differences in the function of the choroid plexus compared to the meninges and its associated arterial vasculature with respect to blood flow, CSF function, neurotoxic insults, and brain trauma.
To begin this protocol place brains just removed from rats immediately after they were euthanized into ice cold, normal saline for five minutes. It is important to allow the brain to cool for this amount of time prior to dissecting so that the four brainin meninges and associated arterial vasculature or MAV can be separated from the surface of the cortex. Next place the brain in the bottom of a glass Petri dish resting on ice with one centimeter of ice, cold, normal saline or 0.1 molar sodium phosphate buffered saline at a pH of 7.4.
All dissection should be done with the brain, mostly immersed in saline. To begin dissection, place the brain dorsal side up in the Petri dish and locate the pineal gland, which is at the most coddle ventral region between the two hemispheres. Just rostral to the cerebellum and just below, or at the surface of the meninges.
Over the colli. Remove the pineal gland using two small bent tip forceps. Note that the pineal gland is pink in color like the cortex, but is often surrounded in remnants of residual blood and vasculature from the brain removal.
Next, flip the brain over upside down in the Petri dish. Then separate the vertebral artery and smaller arteries and meninges covering the pons from the meninges and vasculature of the forebrain. Next, start the removal of the MAV.
It is important to start ventrally in the dissection process with the major arteries of the circle of Willis, middle cerebral arteries and anterior arteries. Next, starting with either hemisphere, use small bent tip forceps to sever the posterior communicating artery just posterior to the internal carotid juncture, thus separating the more anterior and posterior arterial trees of the forebrain. Then repeat the same process for the contralateral hemisphere.
Now gripping the MCA and the anterior artery with the forceps pull gently in an anterior dorsal direction so that the MAV covering the ventral anterior cortex is lifted over and around the olfactory tract. This removes much of the MAV covering the anterior cortex. Turn the brain at a 45 to 90 degree angle to the Petri dish so that the lateral more anterior regions of the MAV surrounding the lateral cortical regions can be freed from the cortex, grip the MCA and associated arteries and gently pull dorsally along the cortex.
If necessary, the ends of the forceps can be used to lift and free the major arteries from the cortex during dissection. Now, remove the more posterior lateral regions of the MAV. Note that it is best to switch from one hemisphere to the other during this harvesting process to ensure that the MAV remains cold and hydrated also switch hemispheres if any resistance in freeing the MAV from the cortex is encountered.
Finally, to remove the most dorsal regions of the MAV surrounding the primary somatosensory and motor cortex, turn the brain dorsal side up to free this section from the brain, use the ends of the forceps along the sagittal sinus. This portion of the harvested MAV can either be kept temporarily in ice cold saline until needed for testing and analysis, or frozen for later processing. Often the MAV covering the most posterior coddle regions of the cortex remains attached.
Its removal is shown in a subsequent section of this video. To remove the choroid plexus first position, the brain dorsal side up and hold it in place with the larger forceps. Then push the smaller forceps down through the midline between the hemispheres and use the ends to puncture through the cortex and corpus callosum into the top of the midline of the hippocampus.
Next, use the forceps to pull the cortex with callosum away from the dorsal hippocampus and septum exposing most of the lateral ventricle. The choroid plexus can be located and identified by the wavy red line demarcating the major artery that runs its length. Use the two ends of the forceps to pry the lateral walls of the third ventricle and enlarge it at its most coddle end.
Then using the small forceps, pull this end of the choroid plexus free. Next, use the forceps to enlarge the very anterior region between the septum and caudate putamen region and pull the choroid plexus free at this end as well. Also perform the same procedure on the contralateral hemisphere to obtain the second remaining choroid plexus.
Again, this tissue can be either kept temporarily in ice, cold, saline, or frozen for later processing. To remove the remaining MAV covering the most posterior coddle regions of the cortex, first, remove the entire hippocampus from both hemispheres exposing the MAV over the thalamus and colliculi. Note that the removal of this portion of the MAV will be much less difficult than its removal from the cortex for the remaining MAV.
By grasping the larger vasculature overlying the anterior portion of the thalamus and gently pulling coddly, this vasculature is connected to the Supra cocurricular network and supplies blood to the dorsal hippocampus and dorsal thalamus network. Pull coddly away and eventually the supra network will be freed and the last portions of the coddle arterial circle can then be reached. At this point, more care must be taken to remove any remaining MAV on the surface of the granular retros lineal primary, auditory, and visual cortices.
Any remaining posterior ventral cortical MAV harvested with this second thalamic and calli MAV section should be removed and added to the first cortical MAV section dissected if they're to be analyzed separately. Next, to compare gene expression profiles between the striatum parietal cortex and MAV regions under control conditions, gene expression analysis can be performed using Agilent 0 1 4 8 7 9 whole rat genome, four by 44, 060 mer oligonucleotide arrays. Further details in processing, scanning and initial data storage can be found in Thomas et al.
When the dissection is executed correctly. The resulting MAV tissues should be in two intact entities weighing about 35 to 45 milligrams total. The initially dissected MAV surrounding most of the cortex should weigh about 25 milligrams, and that covering the thalamus, calli, and occipital cortex should weigh about 15 milligrams.
Tissue should be slightly pinkish in color from the residual blood in the vasculature. The harvested bilateral choroid plexus should be in two intact tissue samples, each weighing one to two milligrams as seen here. Here we see a comparison of gene expression profiles in the striatum parietal cortex and MAV under control conditions.
The top plot shows the expression in the striatum compared to the parietal cortex. While the bottom plot shows expression in the MAV compared to the parietal cortex, it can be seen that the striatum and parietal cortex are much more closely related to each other than the MAV. Here we see the differential gene expression in MAV in response to hyperthermia compared to control, in response to amphetamine compared to control and in hyperthermia versus amphetamine.
Finally, here we see genes with greater than 15 fold expression in MAV compared to parietal cortex and striatum characterized by function. Many of these genes are related to the vascular system and or the immune system. Other genes with very large fold changes are extracellular matrix proteins, saute transporters, and lipid and retinoic acid metabolism.
While attempting this procedure, it is important to remember to be patient while dissecting the MAV. It is also important to have the brain remain submerged in isotonic saline or phosphate buffered saline during the whole dissection. Also, it advise to practice the section of the MAV on four or five rats prior to obtaining tissues for generating experimental data.
