[Sciences/BBB] Endothelial TLR4 and the microbiome drive cerebral cavernous malformations (Tang et al., Nature 2017)

You may have heard about this study that showed how your gut bacteria were responsible for stroke. Of course headline news always love to stretch scientific findings as much as I use to stretch my Stretch Armstrong when I was a kid. However, the paper cited was indeed published in Nature and can be found here:

It is a very interesting paper to read, because a lot of it sounds like a serendipity and lucky strikes. This paper investigated changes in two mouse models of cerebral cavernoma (Ccms). Ccms are a particular type of hemorrhagic stroke because they are mostly genetics (there are three Ccm genes described, in this study they focused on Krit1 and Ccm2) and most of the time go unnoticed. Mutations in those genes result in some alterations in brain microvessels, making some tiny anatomical abnormalities resulting in a higher susceptibility in some of these micro vessels to spontaneously burst and bleed.

The authors of this study have been developing Cre/Lox mice colonies for Ccm2 and Krit1 to better understand the pathology of this disease. The advantage of Cre/Lox is you can knockout a gene in a specific place at a specific time, just by injecting or providing a molecule (usually tamoxifen) that will induce it.

They have been breeding mice that were deficient in Ccm2 or Krit1 and were as expected developing brain micro bleeds (usually around their first two weeks of postnatal age). Following some changes in the animal facility, they observed that a small fraction of their mice colonies suddenly became resistant to cerebral micro bleeds: they still carried the mutations but they fail to develop these microbleeds. Therefore some non-genetic factors were influencing this resistance pattern.
Things became even more interesting as they found that among some of these resistant mice, some developed again the microbleeds within a same littler. The only difference between those developing the microbleeds and those which did not were apparently related to the intraperitoneal (i.p.) injection of tamoxifen. Have the authors provided the tamoxifen through the drinking water, that would have ended the story here.
The authors indeed found that those who reversed their phenotype from resistant to susceptible developed a bacterial infection at the site of the i.p. injection suggesting that such micro bleed was driven by some bacterial factor. They showed that similar results were obtained if they injected LPS (a common Gram-negative antigen) to these mice.

They identified two receptors known to play a role in cellular response to pathogens (we refer such signaling pathways as Pathogen Associated Molecular Patterns or PAMPs): TLR4 (toll-like receptor 4) and CD14 (TLR4 co-receptor). By knocking down these receptors in their Ccm-resistant animals, they were capable to block such bacteria-induced response. The possible interactions of Gram-negative bacteria with these two receptors at the blood-brain barrier maybe enough to trigger the cerebral micro bleeds.

What is also interesting is that mutations in these two genes (some single nucleotide polymorphism or SNP) in patients known to have an history of Ccm also resulted in a higher probability to have brain microbleeds.

I will not spoil the rest of the story but it confirms the presence of a brain-gut axis in Ccm, suggesting the possible effect of the gut microbiota as a risk factor to increased microbleeds in Ccm patients. Let it be clear, these bacteria WILL NOT induce Ccm in normal invididuals. It increase the risk of bleeds in patients already at risk of Ccm.

Another limitation is that in vitro data to confirm the presence of TLR4/CD14 at the BBB and fails to explain how these receptors are triggered by the gut microbiota. The authors suggested a bacteremia (circulating bacteria from gut to the brain via bloodstream) but I remain skeptical about it.

Nevertheless it is a very good paper that worth being read.


[Neurosciences/BBB/Stroke] Breaking The Wall: Brain pericytes impairs the BBB integrity following ischemic stroke injury. 

Interesting study, reminds me one of my earlier study. Brain pericytes are a particular cell type with a disputed origin (some think they come from neural crest, some think they come from mesoderm and some think they are mesenchymal stem-cell like cells) that play an important role in the brain and retinal vascular integrity.
Pericytes play an important function by stabilizing vessels and tightening the blood-brain barrier and the blood-retina barrier. Loss of pericytes have been associated with disruption of the retinal barrier in diabetic retinopathy whereas it was associated with leaky vessels and impaired amyloid beta clearance.
Interestingly, pericytes have been also associated with an impaired barrier recovery following stroke injury acceding to this study by Shih and colleagues (http://www.jneurosci.org/…/2016/11/14/JNEUROSCI.2891-16.2016). The mechanism of action is something known as it involves matrix metalloproteinases (MMPs). MMPs are enzymes that cleaves matrix proteins into fragments as well as tight junction proteins. The increase in MMP activity following ischemic stroke injury was known but until now it was mostly associated with astrocytes and endothelial cells. In my previous work, I have demonstrated that pericytes were deleterious to the barrier function following hypoxic stress.

Source: Rogue Cells Leave the BBB Defenseless During Stroke Trending | Labroots | Virtual Events, Webinars and Videos

[Stroke/BBB] Hyperfibrinolysis increases blood brain barrier permeability by a plasmin and bradykinin-dependent mechanism

This is an article sharing from Oscar, a follower of this page in which he has co-authored (see link to the study below). It is a peer-reviewed article recently published in Blood (a fairly good and robust journal, the official journal of the American Society of Hematology) that focused on the effect of the overexpression of tissue plasminogen activator (tPA) on the BBB integrity.
tPA is the drug of choice for ischemic stroke (80% of total stroke), as it acts as a clot buster (it basically breaks down any clots blocking small brain vessels into pieces). tPA is also until now the only FDA-approved drug. However, tPA has severe downsides. First it has a very limited time-window efficacy that places it uses only for stroke events that have initiated less than 5 hours. Usually this time period is often passed, between the onset of the stroke outcome (the FAST signs), the arrival of 911 and drive to the appropriate stroke center and the differential diagnosis between ischemic and hemorrhagic stroke. After that time-window, tPA becomes pretty useless.
The second issue with tPA is that although it may help reperfuse the ischemic brain tissue and save the neurons inside it during the first hours, it is accompanied by a risk of hemorrhagic transformation within the first 72 hours after stroke by increasing the risk of brain bleeding. Hemorrhagic stroke is even more noxious than ischemic stroke as we have several issues to deal with: a massive entrance of water and ions resulting in brain swelling but foremost the release of hemoglobin (due to the rupture of red blood cells) inside the brain. The heme inside such hemoglobin is a very reactive and aggressive chemical. It is normally inactivated as it transformed into bilirubin and excreted via the bile. But when you have an important release of heme, you have an accumulation of bilirubin (responsible of the jaundice) that has still a toxicity. Therefore, it is important to find a protection mechanism against tPA-induced hemorrhagic stroke injury.
This paper elegantly describe the effect of overexpression of tPA in mice and what does it means in terms of increased plasmin production and its mechanism on action at the BBB. It suggest that plasmin production induced by tPA induces the production of bradykinin (BK), which in turn induces the BBB disruption. Such hypothesis is further supported by the presence of some data obtained from human patients following tPA administration after ischemic stroke injury, in which the authors have measured an elevation of BK levels.
I would say the only drawback of this paper is the absence of in vitro data that directly demonstrate the direct impact of BK on the BBB function and the mechanism by which BK impact the BBB integrity (tight junctions remodeling? destruction of tight junctions by matrix metalloproteinases?).

If you are interested to learn more about this study, you can find the full-text below (behind paywall): http://www.bloodjournal.org/content/early/2016/08/16/blood-2016-03-705384

[Stroke] Surviving stroke injury with a little help of my friends: Astrocytes provides neurons with mitochondria following stroke injury

A very nice study published by Eng Lo (Mass General Hospital), a whiz kid in the field of stroke research. It really brings in a new paradigm in terms of our understanding of stroke injury and stroke repair.
We know that astrocytes play a crucial role in helping the brain recover from stroke injury and try to rescue neurons by secreting growth factors.
But we have been failing to find methods to deliver growth factors in a non-invasive way because producing growth factors by biological engineering is very expensive but also you can only deliver them by directly injecting them in the brain with limited spread.
This very nice piece of work published in Nature and reported by Science (if Science reports on a paper published in a concurrent journal, you bet it should be that good) tells you it is surely a very elegant piece of work here. The paper indeed show that astrocytes seem to go the extra-mile and even provide mitochondria to neurons to help them cope with stroke injury.

Source: Transfer of mitochondria from astrocytes to neurons after stroke : Nature : Nature Research

[Stroke] When a Stroke Patient Gets Worse after Stem Cell Infusions A… : Neurology Today

One of biggest danger facing the stem cell field is the proliferation of “stem cell clinics” inside and outside the United States promising miracle cures for any neurological diseases. The problem with this “stem cell tourism” is not only the huge financial investment into a medical procedure that has yet to filter through scientific rigor and clinical efficacy (at this time, we are still at the stage to assess if these procedures are safe), it is also the risk of developping post-operative complications with “worst case scenarios” happening often, in particular growth of tumor. This is what happened to a stroke patient that not only did not see his stroke injury recovered but now has to fight off the grow of a tumor inside his brain.

Neurology Today discuss the case of this patient, with the intervention of different experts explaining the current state of stem cell clinical trials and the danger of stem cell tourism.

Source: When a Stroke Patient Gets Worse after Stem Cell Infusions A… : Neurology Today

[Neurosciences/Stroke/Stem Cells] A cautionary tale in selling overhyped stroke stem cell therapy

Waking up this morning with a “stunning” finding about the recent publication of a study by Stanford researchers that noted the improved outcome in stroke patients following injection of stem cells have been positively headlined in the fairly serious “Washington Post” journal.
As a neuroscientist and stroke researcher, this sounds like a very good news because there are not much good news when we discuss stroke clinical trials that show something better than placebos. But also me and others like Pr. Paul Knoepfler as he rightly wrote in his blog post to not fail into overhyping and overselling a pilot study and by the way promising the moon to patients and come back to them with a disappointing news.

I thought it would be a great idea to discuss a bit more about this paper, its observations and  outcomes and current limitations.

1. What is the study that was has been cited in the Washington Post and how does it stand in terms of scientific publication?

According to the Washington Post, this study has been published in Stroke journal and authored  by Gary Steinberg, MD-PhD (Stanford University) listed as leading author. Based on the information, we are likely referring to the following article. I have attached a screenshot of the abstract page from the journal website:
First thing, is to classify the authorship ranking. Dr. Gary Steinberg is what we refer as the primary author, usually the person that has performed most of the experiments and analyzed the data. On the other hand, we have Dr. Neil E. Schwartz as a senior author. It is usually the one that has the first thought process, planned the experimental design, wrote and finalized the manuscript and usually the one that have put the money on the table (the funding awardee) to run this study. Here, I would argue that we have a difference in what we consider as the lead author of the paper. I would consider Dr.Schwartz as the lead author due to the ranking, but thats some science bickering.
The paper got published in Stroke, that is the flagship journal of the American Stroke Association (a subdivision of the American Heart Association). Being published in a society journal is a good step but in case not enough to justify the overhyping. Why? Impact factor. Impact factor matters. Stroke, according to the American Heart Association, has an impact factor of 5.76. Thats good but a clinical paper can get better rating. For instance, Circulation (the highest-ranked AHA journal) is listed with an IF~15, whereas Nature Medicine (a mastodon for high-impact translational studies) has an IF of 27.
The paper is surely good, but does it qualify for the “stunning” adjective? Certainly not and the overselling of it is not justified by the publication metric.
Also note the title, this is a Phase I, IIa. So it means it is very a early stage of the clinical trial. Phase I in the first stage of clinical trial in which we test the safety of a novel treatment, Phase IIa is to try if there is any efficacy in a very small subset of patients (less than 50). Again, at this stage, it is a dangerous step to oversell something that yet to show efficacy with hundreds of patients.

2. What does the paper says?
The paper is behind paywall so I cannot publish any figures and text. The paper got one round of revision, as it was received in February 9, revised in April 1 and accepted in April 26. If we consider a 4-6 weeks turnover between the time you submit your draft and the editor respond to you with reviewer comments, we can speculate that the revision was minimal and quickly addressed by the author.
This study rely on using mesenchymal stem cells (MSCs) in a small cohortof patient. The study uses a particular type of MSC, the SB623 bone marrow MSC cell line.
MSCs are a particular type of stem cells. They have the least pluripotency because they have already been engaged inside a differentiation (to make it simple, they are programmed to give rise to blood cells such as white blood cells, red blood cells or platelets) and therefore have little opportunity to be re-wired to form neurons or cardiac cells. However, because they are already into a certain differentiation stage, these cells are considered as the safest for implantation. Other stem cells (such as embryonic or induced pluripotent stem cells) are nefariously known to wreck havoc if injected as undifferentiated (they cause what we call teratomas), safety of differentiated precursor cells (such as neural precursor cells) remains to be addressed. Thats also alleviate the issue encountered with previous stem cell therapy based studies that consisted as injecting a mixture of bone-marrow stem cells without knowing exactly which sub-population is carrying the protective effect. Interestingly, these MSCs carry a plasmid allowing the overexertion of a protein called Notch-1 intracellular domain (ICD). Notch-1 ICD is a fragment of the full Notch-1, that is cleaved by certain enzymes. Such ICD can therefore act as a messenger inside the cell and exert some biological activity.
The study used three doses of cells and were directly injected around the site of infarct (peri-infarct area). In stroke injury, we have the core or infarct area that is considered as the ground zero. We consider it as the necrotic area or the wasteland zone. Everything inside is dead and highly hostile for repopulation. However, the peri-infarct surrounding this zone is battling for days and weeks, torn between signals telling neurons to survive the injury from signals telling neurons to die. This fine balance is one target for therapies as we consider finding factors that can title neurons in favor of survival can help them recover and minimize the loss done by the stroke injury.
What is interesting is that these MSCs have been shown to only survive for one month. Thats a good point for the safety issue.  In this cohort of patients (18 in total), very few side effects were noted suggesting a fairly safe method for up to 12 months. However, one caveat of this study is the lack of proper control or placebo.

3. Why this study has been overhyped and oversold by the WaPo?

All patients improved over the 12 months period compared to their initial timepoint (the day after stroke injury). Furthermore, all three doses have been pooled together, so we cannot tell if there is a better recovery with a higher number of cells. This is a serious concern that has to be mentioned: we cannot tell if these patient recovered by their own or due to the treatment.
If we had a placebo group, we could have been able to compare and contrast the gain due to the stem cell treatment. We also cannot see how each individual and each group have been recovering. It would be interesting to see how age and sex (male/female) played a role in the recovery.
This is the sin of mainstream news media: they have again sinned in overselling a study that is interesting but still lacking solid evidence to sell that case. The study and approach is interesting but the version sold in the news is a  far-stretched version of where the study actual said. Selling it as “stunning” is not only wrong and inappropriate, it is also a dangerous move that will serve some for-profit stem cell clinics to make profits on patients that have been experiencing stroke and despairingly looking for a “miracle cure”.

[Sciences] International Stroke Conference 2016 – Day 2 & 3

As I am writing this post, the #ISC16 is slowly concluding. This is my third ISC (2011 and 2014) and I was glad to see high quality basic science build up over the years.
It was so instructive and intense in terms of science that shut my brain off by the end of the day.
The basic science was very interesting, with a presentation by Dr. James Faber (University of of North Carolina – Chapel Hill) that explored the strain differences between mice in terms of brain vascular collateral branching. The brain is mostly perfused by two main arteries: the anterior cerebral artery (ACA) and the middle cerebral artery (MCA). These are big pipes that branches out into smaller pipes to provide collateral branches, very similar to what you would expect from having faucet water pipes, all being fed by a main water pipe from the city water utility. Dr. Faber nicely described how two different mouse strains (the C57/Bl6 and Balb/c) have opposite collateral densities: one has a lot, the other has almost not. Why it matters? It matters because it can tell us how much the brain tissue is perfused and how well it can sustain ischemia. If you have an auxiliary supply, you are at better odds to cope and recover than if you are lacking it.
Dr. Faber indeed found that one single gene was dictating why these two mice stains show major differences and such differences in branching was only present in the brain but absent in the retina.
Another interesting study was published by Dr. Franklin West (University of Georgia) that have been using pigs as a model for stroke injury and even generating porcine induced pluripotent stem cells (piPSCs) to differentiate into neural stem cells. I found it was a very exciting and interesting aspect as it can help assess the relevance of iPSC-derived model of the BBB by allowing us to directly compare and benchmark iPSC-derived porcine BMECs versus primary porcine BMECs (that can be readily obtainable from pig brains harvested from slaughterhouses).
Aside from scientific presentation, there was also some exhibition mostly focused to clinicians but one key aspect was the development of stroke mobile units as displayed below:

Yep, a CT scan on wheel, specially designed for stroke emergency. As we have mentioned, time matters during stroke injury. However an  important bottleneck in the door-to-operating table is the diagnostic between ischemic and hemorrhagic stroke. On the former, you want to dissolve a clot. On the latter, you want to induce a clot to stop the hemorrhage. This can only be done by doing a CT scan. Thats takes time and by the time you arrive at the hospital and being headed to the radiology, precious minutes have been vanished.
I found that was some impressive advances in technology to have a CT scan on wheel allowing the specialized paramedics to make the diagnosis on the way to the hospital and have the patient directed straight into the operating room. In the evening, we had our poster session and it was an interesting moment to be the senior author, shadowing my student describing the poster:

One sad moment as we were un mounting the poster was to see the poster graveyard were orphaned poster are left abandoned by their owners, like pets on the roadside during holidays. Unfortunately, no poster shelters for these poor folks. I always consider that posters should be brought back to the lab to display your lab achievement and only die due to aging (outdated data).

As the conference ending up, there are some special lecture. The Willis Award was given this year to Dr. Ulrich Dinargl (Center for Stroke Research, Berlin), also co-editor of the Journal of Cerebral Blood Flow & Metabolism. Dr. Dinargl nicely advocated for preclinical models of stroke, as we learn from failures and from negative experiences. The recent move of several journals, including JCBFM, to have a “negative results” section is a welcome move than can save valuable time and money to other research groups and avoid others to engage into scientific dead-ends.

Auld Lang Syne, as the curtain drops, we are already eyeing on next year……with a much closer venue. Glad to see the next ISC being held in Houston, Texas.