This work was funded by NCTR/FDA.

Although not shown in the video, rats were first given an overdose of 300 mg/kg of pentobarbital, resulting in anesthesia in less than 3 min, and then killed by decapitation. Their brains were then rapidly but carefully removed from the skull and chilled in ice-cold normal saline for 5 min. It is important to let the brain cool for this amount of time prior to dissecting the MAV so that the MAV can be separated from the surface of the cortex (top of layer I). The brain was then placed in the bottom of a glass Petri dish resting on ice that was full (1 cm deep) of ice-cold normal saline or 0.1 M sodium phosphate-buffered saline pH=7.4. All the dissection is done with the brain for the most part immersed in saline.
1. Removal of the Pineal Gland
<ol>
	<li>Place the brain dorsal side up (relative to the bottom of the Petri dish). The pineal gland is located at the most caudal ventral region between the two hemispheres just rostral to the cerebellum (pineal recess), and is just below or at the surface of the meninges over the colliculi. Remove the pineal gland first using two small bent tip forceps. It is pink in color like the cortex but is often surrounded in remnants of blood and vasculature residual from brain removal.</li>
</ol>
2. Removal of the MAV
<ol>
	<li>Flip the brain over "upside down" and separate the vertebral artery, smaller arteries and meninges covering the pons from the meninges and vasculature of the forebrain. Removal of the forebrain MAV is then started. 
	It is important to start ventrally in the dissection process. The large major arteries of the circle of Willis, middle cerebral arteries (MCA) and anterior arteries are the strongest and serve as "scaffolding" by which the smaller superficial arteries and arterioles of the pial membrane can then be removed from the cortex with the remainder of the meninges. See review by Scremin7 for more visual representations and details of the cerebral surface vasculature.</li>
	<li>Starting with either hemisphere, use the 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. Repeat the same process for the contralateral hemisphere.</li>
	<li>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 (piriform, olfactory tubercle) is lifted over and around the olfactory tract. This removes much of the MAV covering the anterior cortex (cingulated and orbital).</li>
	<li>Turn the brain at a 45 to 90&deg; angle to the Petri dish. The lateral, more anterior regions of the MAV surrounding the lateral cortical regions (granular insular, secondary somatosensory and auditory cortex) can be freed from the cortex by gripping the MCA and associated arteries and gently pulling 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.</li>
	<li>Now remove the more posterior lateral regions of the MAV. (Note, 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, if resistance in freeing the MAV from the cortex is encountered on one hemisphere switch to the contralateral hemisphere temporarily and then go back to the original hemisphere later.)</li>
	<li>To remove the most dorsal regions of the MAV surrounding the primary somatosensory and motor cortex turn the brain dorsal side up (relative to the bottom of the Petri dish). To finally free the MAV from brain, use the ends of the forceps along the sagital sinus. 
	This portion of the harvested MAV can be either kept temporarily in ice cold saline until testing and analysis or frozen for later processing. Often the MAV covering the most posterior/caudal regions of the cortex (entorhinal, visual and most caudal auditory cortices) remains attached to the cortex. Its removal is shown later in the video when the MAV between the retrosplenial cortex and overlying the colliculi are dissected.</li>
</ol>
3. Removal of the Choroid Plexus
<ol>
	<li>Position the brain dorsal side up and hold in place with the larger forceps Push the smaller forceps down through the midline between the hemispheres and then use the ends to puncture through the cortex and corpus callosum (&asymp;-3.3 mm from bregma) into the top of the midline of the hippocampus.</li>
	<li>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 the most caudal end (&asymp;-4.3 mm from bregma8).</li>
	<li>Using the small forceps, pull this end of the choroid plexus free. Then go to the very anterior region between the septum and caudate/putamen (most rostral extent, &asymp;+1.6 mm from Bregma) and with the forceps enlarge this section of the ventricle and pull the choroid plexus free. 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 until testing and analysis or frozen for later processing.</li>
</ol>
4. Removal of the Remaining MAV in the 3rd Ventricle Overlying the Thalamus and the Colliculi as well as that on the more Caudal Regions of the Cortex including Retrosplenial, Auditory and Visual Cortex
<ol>
	<li>Remove the entire hippocampus from both hemispheres exposing the MAV over the thalamus and colliculi. The removal of this portion of the MAV is much less difficult than the removal of MAV over the cortex.</li>
	<li>Pull this portion of the MAV free by grasping the larger vasculature overlying the anterior portion of the thalamus and gently pulling caudally. This vasculature is connected to the supra collicular network and supplies blood to the dorsal hippocampus and dorsal thalamus network. Pull it away caudally and gradually free the supracollicular network until reaching the last portions of the caudal arterial circle vasculature. 
	At this point, more care must be taken to remove any remaining posterior ventral MAV on the surface of the granular retrosplenial, primary auditory and visual cortices. As with the other section of MAV covering the more anterior portions of the cortex, this tissue can be either kept temporarily in ice cold saline until testing and analysis or frozen for later processing. Any remaining posterior ventral MAV harvested with this second MAV section should be removed and added to the first dissected MAV section covering the cortex if they are to be analyzed separately.</li>
</ol>
5. Representative Results
When the dissection is executed correctly, the resulting MAV tissues should be in two intact entities weighing about 35 to 45 mg total. The initially dissected MAV surrounding most of the cortex should weigh about 25 mg and the MAV covering the thalamus, colliculi and occipital cortex should weigh about 15 mg. It should be slightly pinkish in color from the residual blood in the vasculature. The bilateral choroid plexus harvested should be in two intact tissue samples with each weighing 1 to 2 mg. Although excess water can be removed from the MAV prior to storage or processing using tissue paper such as that used in lens cleaning, to avoid tissue loss it is not a good idea to remove excess water from the dissected choroid plexus. Figure 1 was generated for comparing expression profiles in three regions (striatum, parietal cortex and MAV) under control conditions using Agilent-014879 Whole Rat Genome 4x44K 60mer oligonucleotide arrays (G4131F, Agilent Technologies, Palo Alto, CA; NCBI GEO Accession # GPL7294; GSE23093 and GSE29733 that are present at the NCBI GEO repository). The two plots in the figure pictorially show the expression in 1) the striatum compared to the parietal cortex and 2) the MAV compared to the parietal cortex. It is clear that the striatum and parietal cortex are much more closely related to each other than the MAV. The data comparing the choroid plexus with the MAV will be presented in a future publication. However, it is likely that the expression profiles in the MAV and choroid plexus are more closely related to each other than they are to the striatum and parietal cortex. Figure 2 shows the differential gene expression response to hyperthermia (EIH) versus AMPH in MAV compared to control and each other.
The gene expression of more than 11,000 genes with official NCBI gene symbols in MAV of control animals was compared to those recorded in the striatum and parietal cortex. Over 2000 of these genes had a 2.5-fold or more differing expression in the MAV compared to the two neuronal-rich tissues, and 550 genes had a 10-fold greater difference in expression. Slightly more than 40% (253) of these were genes with a decreased expression of 10-fold or more in MAV and were related to neuronal function. There were 343 genes with over a 10-fold or greater expression in MAV compared to parietal cortex and striatum. This list of genes was used as a starting point with which to determine genes with a very high enrichment in the MAV. Table 1 lists the genes with greater than 15-fold expression in MAV compared to parietal cortex and striatum and categorizes them with respect to function. Many of these genes were related to the vascular system and/or the immune system. These types of genes should be enriched about 5- to 10- fold to because of the increased vasculature itself and blood present within it. However, even for genes with known general vascular functions, an increase above 15-fold indicates enrichment in MAV. There were also numbers of genes with very large fold changes that were; 1) extracellular matrix proteins (not specifically related to vasculature), 2) solute transporters and 3) lipid and retinoic acid metabolism. As well, few growth and differentiation gene or transcription factors were found.
<img alt="Figure 1" src="/files/ftp_upload/4285/4285fig1.jpg" /> 
Figure 1. Comparison of the Gene Expression in MAV with Parietal Cortex and Striatum. Volcano plot comparing the gene expression levels of the parietal cortex and striatum (top plot) and the parietal cortex and MAV (bottom plot). The red symbols denote the genes that are significantly different between regions at p &lt;0.05 and have a 1.5-fold or greater differential expression between regions. The black symbols represent genes which do not significantly differ between regions (p &gt;0.05) and are less than 1.5-fold differing in expression. The pink symbols indicate genes with less than a 1.5-fold difference in expression but statistically significantly different at p&lt;0.05. The yellow symbols identify genes which have a 1.5-fold or greater expression but this change is not statistically significant at p&lt;0.05.
Abbreviations
AMPH amphetamine 
EIH environmentally-induced hyperthermia 
MAV meninges and associated cerebral vasculature 
Norm normothermic controls
<img alt="Figure 2" src="/files/ftp_upload/4285/4285fig2.jpg" /> 
Figure 2. Effects of hyperthermia and amphetamine on gene expression in MAV. Volcano plots comparing gene expression levels in the MAV after saline, EIH or AMPH at the 3 hr time point. The red symbols denote the genes that are deemed to be significantly different while the black dots/circles represent genes which do not significantly differ between regions. Additional statistical analysis was used to verify which expression differences between control and treatments were in fact statistically significant.
<table border="1">
	<tbody>
		<tr>
			<td fo:width="1in">MAV Expression Brain Expression</td>
			<td fo:width="4.5in">NCBI Gene Symbols</td>
			<td fo:width="1.5in">Tissue Specificity or Reported Function(s)</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Anxa2a, Col8a1, Des, Esm1, Glycam1, Kla, Lect1, Lepr, Lum, Myh11, Ogn, Tagln, Thbs2, Tnnt2 </td>
			<td>Vasculature &amp; Heart</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Ccl19, Cd74a, Defb1, Lrrc21, Mgl1, Mrc1, Msln, Pla2g5, Prg4, RT1-Bb, RT1-Da</td>
			<td>Immune System</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Adh1, Aebp1, Aldh1a2, Gstm2 </td>
			<td>Retinoic Acid &amp; Lipid Processing</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Cdh1, Col3a1, Col1a2, Colec12, Cpxm2, Cpz, Dcn, Emp3, Gpc3, Nupr1, Omd, Pcolce Slamf9, Tmem27, Tspan8 </td>
			<td>Extracellular Matrix &amp; cell-cell junctions</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Aqp1, Asgr1, Kcnj13, Slc5a5, Slc6a13, Slc6a20, Slc22a6, Sned1, Ttr </td>
			<td>Ion &amp; Solute Homeostasis</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Alx3, Cdkn1c, Foxc2, Igfbp2, Ifitm1, Ifitm2, Nkx6-1, Osr1, Prrx2, Sfrp1, Tbx15. Upk1b, Wisp2, Wnt6</td>
			<td>Development &amp; Transcription Regulation</td>
		</tr>
		<tr>
			<td>&gt; 50-fold</td>
			<td>Gpha2, Mfap5, Mpzl2 Plac8, Scgb1c1, Sostdc1, Steap1</td>
			<td>Unknown &amp; Miscellaneous</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Anxa1a , Angpt2, C6a, Gjb2, Ptgis, Thbd, Timp1</td>
			<td>Vasculature &amp; Heart</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Casp12, Ccl2, Ccr1, Cd14, Cxcl10, Ifitm3, Klra5, Lgals1, Lgals3, Ms4a4a, Ms4a7, Plscr1, Spp1, Xcl1</td>
			<td>Immune System</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Col6a3, Cpxm1, Efemp1, Fmod, Mgp, Nid2</td>
			<td>Extracellular Matrix &amp; cell-cell junctions</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Cp, Cubn, Gcgr, S100a6, Scn7a, Sct, Slc4a5, Slc16a11, Slco1a5</td>
			<td>Ion &amp; Solute homeostasis</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Cfd, Ch25h, Crabp2, Rarres2</td>
			<td>Retinoic Acid &amp; Lipid Processing</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Bmp6, Casp12, Dab2, Folr1, Igf2, Msx1, Tbx18, Twist1, Wnt5b</td>
			<td>Development &amp; Transcription Regulation</td>
		</tr>
		<tr>
			<td>30 to 50-fold</td>
			<td>Cela3b, Cln6, C1qtnf7, Copz2, Dhrs7c, Gng11, Prss23, Srpx, Vim</td>
			<td>Unknown &amp; Miscellaneous</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Adm, Angpt1, Angptl2, Bgn, Cklfa, Cnn1,Cox8h, Ctsk, F13a1, Gja5, Klf4, Lox, Lyz, Myl9, Procr, Pros1, RT1-Ba, Serpinb10, Serping1, Serpinf1, Tgm2, Tnmd, Trim63, Txnipa, Vamp5, Vtn</td>
			<td>Vasculature &amp; Heart</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Adaa, Bst2, Ccl6, Cd40, Cd68, Ctsc, Dap, Faim3, Fkbp9, Fxyd5, Fmo1, Glipr1, Ier3, Ifi47, Igsf6, Msc, Nfatc4, RT1-Db1, Serpinb1a, Tir4, Tubb6</td>
			<td>Immune System</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Adamtsl4, Col1a1, Crb3, Dpt, Egfl3, Fbn1, Fbln1, Fbln5, Fn1, Itgb4, Lama2, Lgals3bp, Loxl1, Mfap4, Mmp14, Mmp23, Ppic, Serpinh1, Timp3</td>
			<td>Extracellular Matrix &amp; cell-cell junctions</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Cybrd1, Selenbp1, Slc2a4, Slc9a2, Slc13a3, Slc16a4, Slc22a8, Slc22a18 </td>
			<td>Ion &amp; Solute homeostasis</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Agpat2, Bdh2, Cyp26b1, Lpar3, Ltb4dh, Olr1, Pon3, Rbp1, Rbp4</td>
			<td>Retinoic Acid &amp; Lipid Processing</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Atf3, Dkk4, Eya2, Ifitm7, Ltbp1, Mustn1, Nr2f2, Ptrf, Sphk1, Tgfbi, Tcea3 Tcfap2b, Wnt2b, Wnt5a, Zic1</td>
			<td>Development &amp; Transcription Regulation</td>
		</tr>
		<tr>
			<td>15 to 30-fold</td>
			<td>Adamts12, Adamtsl3, C1r, Crispld2, Crygn, Cyp1b1, Dse, Enpep, Enpp2, Epn3, Flna, Fmo3, Gnmt, Gprc5c, Hspb1, Klc3, Krt18, Krt19, Mdk, Mesp1, Ms4a6a, Ms4a6b, Ms4a11, Net1, Pdlim2, Phactr2, Plp2, Ppp1r3b, Pqlc3, Rin3, Spag11, Sult1a1, Tmem106a, Wfikkn2</td>
			<td>Unknown &amp; Miscellaneous</td>
		</tr>
	</tbody>
</table>
*Genes included in the table must be both more than 15-fold above expression in the striatum and parietal cortex and 5-fold above the background levels. The lower of the two ratios (MAV/striatum or MAV/parietal cortex) for each gene was used for grouping.
aGenes are found in endothelial cells but also likely play a major role in mediating immune responses.
Table 1. Genes with a 15-fold* or more increased expression in MAV compared to striatum and parietal cortex.

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Care. 10, 295-305 (2009).</li><li>Ohta, K., 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=Delayed+ischemic+stroke+associated+with+methamphetamine+use.">Delayed ischemic stroke associated with methamphetamine use.</a> J. Emerg. Med. 28, 165-167 (2005).</li><li>Brenner, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropsychological+and+neuroimaging+findings+in+traumatic+brain+injury+and+post-traumatic+stress+disorder.">Neuropsychological and neuroimaging findings in traumatic brain injury and post-traumatic stress disorder.</a> Dialogues Clin. Neurosci. 13, 311-323 (2011).</li><li>Case, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflicted+traumatic+brain+injury+in+infants+and+young+children.">Inflicted traumatic brain injury in infants and young children.</a> Brain Pathol. 18, 571-582 (2008).</li><li>Maas, A. I., 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=Prognostic+value+of+computerized+tomography+scan+characteristics+in+traumatic+brain+injury:+results+from+the+IMPACT+study.">Prognostic value of computerized tomography scan characteristics in traumatic brain injury: results from the IMPACT study.</a> J. Neurotrauma. 24, 303-314 (2007).</li></ol>

We have nothing to disclose. The work presented here may not necessarily represent the views of the Food and Drug Administration.

Technical aspects of MAV and choroid plexus dissection
It is critical during the dissection of the MAV to have the brain "submerged" most of the time in the normal saline or sodium phosphate-buffered saline. Allowing the MAV and cortex to dehydrate during dissection will result in the MAV adhering tightly to cortical layer I. This will increase the amount of cortical tissue harvested with the MAV and/or result in the inability to remove these adhering MAV sections from the cortex. Also, patience is necessary during dissection. Again, if one encounters some resistance in removing the MAV from the cortex in one hemisphere during dissection, switch to the other hemisphere and go back later to the side where resistance was encountered. This method does take some practice to perfect and requires about 5 to 15 "practice" dissections before a consistently uniform harvest of MAV tissue is achieved. The method of starting the dissection and removal of the MAV at the arterial circle at the base of the brain facilitates the removal of the meninges by using the larger vasculature as support "scaffolding."
Much of the blood originally in the MAV vasculature after sacrifice should be ejected during the dissection, and less than 5% of the RNA harvested from MAV should be of blood origin. It is possible to remove almost all the blood by perfusing the animal with 50 to 70 ml of saline, using histological techniques, prior to decapitation and brain removal. However, it is then much more difficult to visualize the MAV tissues during dissection. The three most likely sources of "contamination" of the MAV tissues are the pineal gland, cortical layer I tissue and residual olfactory bulb tissue but it is also possible to get pituitary gland tissue in the preparation. Pineal contamination in the MAV is detected by the MAV containing a round 1 to 2 mm diameter sphere that is colored dark red. The primary function of the pineal gland is normally considered to be completely different from the MAV. Its primary function is to produce melatonin, which regulates aspects of the circadian rhythm9, and in lipoxygenation10 of lipid precursors. Although the gene Lox, which regulates lipoxygenase activity, was thought to be present in adults primarily in the pineal gland10, our results indicate that it is also consistently present at significant levels in MAV. Residual cortical or olfactory bulb contamination results in the pinkish web-like MAV tissue having some 1 to 2 mm denser tissues that are much paler in color enclosed in it. This can be spotted by "floating" the MAV on the surface of the buffer, and then removing the heavier cortical or bulb tissues using the two dissecting forceps.
If contamination of the MAV from cortical layer I tissue, hypothalamic tissue or the pineal gland is present, then several genes will have a significantly greater expression than what is normally present in a "clean" dissection. Contamination of the MAV with pineal gland will result in higher expression of arylalkylamine N-acetyltransferase (Aanat) which is necessary for melatonin synthesis. Unfortunately, if pineal contamination occurs during MAV dissection, the levels of Lox in MAV will not be able to be accurately determined. Contamination of the MAV with cortical tissue will result in higher expression of neuron-specific genes such as hippocalcin (Hpca), parvalbumin (Pvalb) and/or neuronal pentraxin receptor (Nptxr). Contamination of MAV with hypothalamic tissue will result in the higher expression of growth hormone 1 (Gh1), proopiomelanocortin (Pomc). In the event that some contamination does exist in the harvested tissues, most of these genes can be identified and not used in gene expression analysis. This is particularly important for genes present in the circulating blood that may still be present in MAV or choroid plexus.
Interpretting gene expression data from harvested MAV and choroid plexus
The cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis4,11 ,12 ,13 ,14 ,15 ,16 ,17 . The tissue harvest is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia1,2 through gene expression analysis. These results are commensurate with what is also known about severe hyperthermia and exposure to amphetamines to the brain vasculature in general 18,19,20 . Human clinical data supports the belief that the subdural vasculature is sensitive to damage by amphetamine and methamphetamine 21,22 . As well, the major and minor cerebral vasculatures including those in the pial layer of the meninges that are harvested in MAV are of potentially high interest when investigating concussive types of head trauma 23,24 25 . The current gene expression profile yielded from the MAV indicates that the pial and possibly the arachnoid meningeal membranes may play a greater role than expected in regulating CSF composition. In the future, through the use of dissecting microscope techniques, it may be possible to further separate the tissues comprising the MAV so that the pial and arachnoid membranes can be separated from the larger arterial vasculature present and possibly from each other. This will enable a better interpretation of the function of each of these 3 components of the harvested MAV.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Normal Saline (9 grams saline/ 1 Liter H2O)</td>
			<td>In-house</td>
			<td>&nbsp;</td>
			<td>Dissection buffer</td>
		</tr>
		<tr>
			<td>0.1 M Sodium Phosphate pH 7.4</td>
			<td>In-house</td>
			<td>&nbsp;</td>
			<td>Dissection buffer</td>
		</tr>
		<tr>
			<td>Bone Rongeurs</td>
			<td>Roboz</td>
			<td>RS-8300</td>
			<td>Skull bone removal</td>
		</tr>
		<tr>
			<td>Forceps-bent tip (4" long &amp; 0.8 mm wide tip)</td>
			<td>Roboz</td>
			<td>RS-5135 or RS-5137</td>
			<td>Dissecting forceps</td>
		</tr>
		<tr>
			<td>Forceps-straight tip (5.5" long )</td>
			<td>Roboz</td>
			<td>RS-8124 or RS-8104</td>
			<td>Thumb Dressing forceps</td>
		</tr>
	</tbody>
</table>

a visual description of the dissection of the cerebral surface vasculature and associated meninges and the choroid plexus from rat brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

Watch this Scientific Journal Video about A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain at JoVE.com

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

a-visual-description-dissection-cerebral-surface-vasculature

Research

JoVE Journal

Neuroscience

47.9K Views.  National Center for Toxicological Research. This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

Video: A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

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 ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...