Hello, My name is Julie Saugstad, and I'm from the Oregon Health and Science University in Portland, Oregon, that's located on the west coast of the United States. And here I'm going to tell you about the collection and processing of cerebrospinal fluid for extra side. Cerebrospinal fluid, or CSF, is a clear transparent fluid that's drive from plasma. It's made in the ventricular system, a set of four interconnected brain regions containing the choroid plexus, a specialist tissue involved in the production of CSF. Adult humans make about 500 mils of CSF each day, and there's typically 100 250 mils of CSF present at any one time. CSF flows from the lateral ventricles at the top of the image, down to the third and then the fourth ventricle, and then they go to the central canal of the spinal cord. The ventricular system and the canal are aligned with the ependyma, a type of neurogia and modified tight junctions between these dependable cells can control fluid released across the epithelium, and this allows for very free exchange between CSF, brain tissue, and the spinal cord. So we know that CSF bathes the brain and the spinal cord, and thus it's the optimal bio fluid to actually study central nervous system diseases. CSF has a number of functions, it can protect the brain and the spinal cord from injury by acting as a fluid cushion and a mechanical berry against shock, it can provide buoyancy. The mass of the human brain is approximately 1400 grams, but suspended in CSF, the net weight of the brain Is equivalent to about 25 grams. CSF can clear metabolic waste, including moving antibodies, chemicals and pathological products of disease away from the brain and the spinal cord and into the bloodstream where they can be cleared. CSF also transports extra sire components to remote sites in the brain, for example, hormones or hormone releasing factors that can then exert their effects on brain cells. And now we know that CSF contains extra site or vesicles, these include exorcisms and micro testicles, and they also contain RNA bound to lipoprotein particles that can then function in cell to cell communication. There's a nice paper that really highlighted a number of roles for EVs in the healthy CNS. These include oligoedrocytes-derived EVs that could be taken up by an act of microglia. We know that Ev is released from microglia can promote our transmission. We also know that in our own activity can trigger EV release from all of the dangerous sites. The astrociytes-derived EVs can actually aid in angiogenesis, however, EVs can also be involved in CNS disorders. It's been shown that neural EVs contain HIV tap protein, aggregated town named loydbeta, which are both involved in Alzheimer's disease, and Alpha's nucleus, which is involved in Parkinson's disease. It's been shown that microglia dry DVs contain pro inflammatory cytokines and that you're only EVs can transfer aggregated proteins into different cells. We also know that oligodendrocytes transfer EVs containing molecules that can activate microglia. And additionally, astrocyte EVs can impair neuronal function. So these studies all tell us together that, CSF EVs cannot only contribute to healthy CNS functions, but also to a number of CNS disorders. CSF is one of the bio fluids that can be collected from living donors, and one of the ways to do that is by a lumbar puncture or spinal tap. Years ago, consortiums such as the NIH funded Alzheimer's disease centers, established standardized protocols for lumbar CSF collection. And this was to ensure consistency in the collection and processing of CSF, and that allows direct comparisons of study results from labs across institutions. While the number of the maximum volume of CSF that can be collected from one patient at one visit is 20 to 25 mils, some recommend collecting smaller amounts of CSF, and this is to avoid changes in the gradient of CSF components. One major drawback to lumbar puncture, is an invasive procedure, and short term temporal collections are difficult to obtain. CSF can be also obtained from central sites such as surgical access to ventricles or the subarachnoid space. The advantages that large amounts of CSF can be collected and that if there's a shunned in place, temporal collections can be taken at distinct times after access. The drawback is the CSF here likely contains blood as well as damage cells from surgical access to the brain. Because of the differences in the collection sites, lumbar punctures definitely thought of as a cleaner CSF sample. So that said, CSF collection sites should be consistent within a study as the differences in their content may or will influence downstream study outcomes. This is destroyed introduction just to show you how lumbar CSF is collected. This is typically done in the morning under fasting conditions, and the donor is lying on their side, are seated and then a site in the lower back is sterilized and injected with a local anesthetic. Then a small 24 gauge Sporotte spinal needle is inserted into the spinal sack either between L3- L4 or L4-L5. The first 3-5 minutes collected is sent for clinical analysis typically, and it may contain a bit of blood from where the needle went into the spine. At this time, five mills cereal syringes of CSF were collected, and then they're mixed. The CSF is then divided into aliquots, snap-frozen on dry ice and stored at -80°C. The tubes of aliqouts are numbered to account for any gradient effect in the studies, and they're also given a de identified subject number with no personal information. And this is to facilitate later collaborations and sharing of samples between sites. Now that we know that CSF and other bio fluids contain EVs, the recommendation going forward is that the CSF should be centrifuge at a low speed, say 2000 to 4000 g force to remove any cells or degrees from the CSF before storage at -80°C. So once the CSF is collected, there's a number of clinical measures that can be done to assess the state of the patient at the time CSF was collected. These include changes in pressure if there's increased pressure, would indicate that there is a sign of trauma of a stroke of hydrocephalus. Things that the clinics that will follow up on with the patient, if there's decreased pressure, it would suggest that there is a leak of CSF into another body cavity, resulting either from a lumbar puncture or medical procedures involving the brain or the spinal cord. If there's an increase in protein in the CSF, it would indicate that there's either blood in the CSF or that the patient has diabetes, a potential tumor and injury, or some type of inflammation or infection. If there's a decrease in glucose, it would suggest they have hyperglycemia or it could be that the patient has tuberculosis or meningitis infection due to bacteria, viruses or fungus is CSF typically contains no red blood cells. But if there's an increase in red blood cells, it will show you that there's bleeding into the spinal fluid and again resulting from a traumatic lumbar puncture or some type of brain injury. There's also very few white cells in the CSF in a normal patient. But if there's an increase in white blood cells, it would indicate that the patient has meningitis, a potentially at the beginning of a chronic illness, et cetera. So these would all lead a clinician to move forward and do additional tests. While not ready for prime time yet, there is potential for changes in CSF EVs to serve as clinical measures for CNS diseases in the future. This slide is here to show that CSF and serum are actually pretty similar in a number of substances as listed in the table, and their amounts are about equivalent between the two bio fluids. However, the one major difference is in the protein content, serum has about 200 times more protein than does CSF. Those of you got a small amount of blood and CSF, it could greatly affect downstream assays, particularly if you're looking for protein expression in the CSF. Earlier, I mentioned studies that we've done to examine the difference between lumbar and central CSF measures. This was a collaborative study that we did a few years ago, and the image here shows that by nanoparticle tracking analysis, there are far more nanoparticles detected by NTA in central CSF than in lumbar CSF. Consistent with that, we also see a large increase in the amount of total RNA in central CSF versus that from lumbar CSF. There's about 10 to 50 times more RNA in central CSF. S these studies show that lumbar CSF is probably cleaner, and it contains a lower number of extra cellular testicles or nanoparticles and less total RNA than does central CSF. And it's likely because there's more contaminants that may show up in central CSF due to damage during access to the brain. This is a very nice study to look at the effect of age on CSF EVs on panel A, you can see that the size of EVs doesn't change in different time periods, of age, whether it's an infant, a teenager or anybody over 70. However, in panel B, you can see is that you go from an intent to an older person that the average number of CSF EVs does decline with age. So it's really important if you're looking at CSF EVs studies that you match the age of the study participants to account for any changes in the number of EVs over time. Other modules are going to be discussing EV isolation methods. But I just want to point out that in the beginning of the EV isolations, a lot of people use ultracentrifugation and then potentially followed by density gradients so that you could get pure EVs. One of the problems with this method is that it's not amenable to smaller volumes of samples such as what you would get with a lumbar CSF sample. So for lumbar CSF sample, we actually like to use size exclusion chromatography. This is because this is the SEC is more gentle on the EVs, but also that the smaller columns can easily separate out micro testicles from EVs and then there's a little bit of overlap between EVs and the proteins that come out later from the CSF. However, it's the cleanest way we've found to be able to isolate CSF EVs. So for isolation methods, we prefer to use SEC because it's more gentle on EVs, it doesn't use any harsh free agents. And it's amenable for isolating EVs from small starting volumes of lumbar CSF samples, such as half a milliller that you would get from a bio bank. In summary, what I've conveyed to you today is that the CSF based the brain and the spinal cord, and it's an optimal biofuel to study CNS diseases. It contains EVs, including exorcisms and microvesicles, and what we didn't talk much about today. It also contains RNA bound of lipoprotein particles, and these can all function and cell to cell communication. In that regard, CSF EVs can contribute to healthy CNS function as well as the CNS disorders, and again, the CSF collection site should be consistent within a study. That's because differences in lumbar versus central CSF will influence downstream outcomes. And then for CSF EV studies, we suggest that the CSF should be centrifuge at a low speed 2 to 4000 g to remove any cells or debris from the CSF before storage at -80. And again, we're not ready for prime time, we believe there's potential for changes in CSF EVs to surface clinical measures for CNS diseases and to remind you, serum has about 200 times more protein than does CSF. So even if you have a small amount of blood in the CSF, it can greatly affect downstream essays, particularly figure looking for proteins or proteins on EVs. In that vein, lumbar CSF also has less eaves and nanoparticles and less total RNA than the central CSF. And finally, we prefer to use SEC for isolation of EVs, it's more gentle. It uses no harsh re agents, and it's amenable for isolating EVs from small starting volumes of lumbar CSF, such as which you would get from a bio bank. So in closing, I've added a bit of additional reading if you're interested. One is on the bio banking of cerebral spinal fluid, and the other is on the cerebrospinal fluid and blood biomarkers. This is a very comprehensive paper, but in fact it's got a large amount of information about CSF itself if you're interested in. So for that, I'd like to thank you for listening today, I hope you've learned something about CSF and EVs, and thank you again.
