Categories
Neurosciences Pharmacology

[Sciences/Pharmacology] Death by Benadryl Tik-Tok challenge: making the case on an interesting neurotransmitter

Another day, another “dose makes the poison” day. This time, it is about diphenyhydramine (Benadryl(R)). You surely heard about the recent “Tik-Tok challenge” launched by some folks that is basically overdosing on Benadryl(R) with some report of death as reported here. So far, we have 3 teenagers in Texas that had to be hospitalized following overdose on Benadryl (up to 14 doses at once) and one death on Oklahoma (dose unreported). Diphenyhydramine is usually taken as an anti-allergic due to its anti-histamine activity (which is a major molecule released by basophile white blood cells, responsible for the allergic response). Interestingly, the reason these kids took Benadryl(R) was not for a major allergic reaction to pollen or animals hair. But apparently “to get high”. This raised me some questions as histamine is not a major neurotransmitter as glutamate, or dopamine are.

This raised my curiosity about the role of histamine in the central nervous system (CNS) and how would diphenylhydramine come to play? As usual I love to start with the chemical structures. Histamine is on the left, diphenyhydramine is on the right:

As you can see, histamine is not too far from histidine, an aminoacid. The only thing missing is the carboxyl group (-COOH) on the carbon alpha. Diphenyhydramine that I will call DPH to ease the typing) has not much anything in common with histamine.
Histamine is not a common neurotransmitter, and indeed has a very specific nucleus, according to Haas and Panula (https://pubmed.ncbi.nlm.nih.gov/12563283/), located in the tuberomammary nucleus, which appears located between the pons and the thalamus, likely part of the hypothalamus. As other nuclei, the histaminergic system is made by projections towards various region of the brain as represented below:

We can see projection into various region including the striatum/substantia nigra (which is involved in movements execution and affected in Parkinson’s disease), cerebellum (involved in the gait posture and coordination in movements like walking), hippocampus (memory formation) or amygdala (which deals with various things including pleasure). What is more interesting is the presence of projection into the medulla, which means it can likely modulate some vegetative functions including breathing or hearbeat regulation.
What is interesting is that such histaminergic system appears well conserved in evolution. We found in mollusk and we found it in mammals, which is interesting. It also has 3 major receptors in the brains (named H1R, H2R and H3R respectively). The biological functions of histamine appears various and include function in the wake/sleep cycle, inhibitor of neural function (which is important as we discuss DPH pharmacology), feeding behavior, fluid intake regulation, thermoregulation and others. But what is interesting is the ability of histamine to act as a hedonist molecules, including impaired reward behavior and altered cognitive functions when volunteers were given H1-antihistamines.

This brings us to the pharmacology of anti-histamines. Interestingly, the first generation of anti-histamines was marked by their persistent side effects on the central nervous system (CNS) and included DPH. These first-generation of drugs side effects were somnolence (a common side effect reported with Benadryl), drowsiness, lack of concentration and attention. The reason why such side effects occur is because these compounds have a very good blood-brain barrier (BBB) permeability, which can exert their central effects easily. To remediate with such issue, a second-generation developed in the aim of reduced BBB permeability was developed such as fexofenadine (Allegra(R)) which is commonly sold as “non-drowsy” anti-histamine.

Now if you look at the Lexicomp (which is a drug database pharmacists commonly access to obtain a detailed drug information), there is an important warning on Benadryl(R): “CNS depression: May cause CNS depression, which may impair physical or mental abilities; patients must be cautioned about performing tasks which require mental alertness (eg, operating machinery or driving).”. If we dig in further we can see two major adverse effects reported:
Cardiovascular: Chest tightness, extrasystoles, hypotension, palpitations, tachycardia
Central nervous system: Ataxia, chills, confusion, dizziness, drowsiness, euphoria, excitement, fatigue, headache, irritability, nervousness, neuritis, paradoxical excitation, paresthesia, restlessness, sedation, seizure, vertigo

There is a serious risk on the cardiac side, whereas we can see that on the CNS side we have some effects sought as it use for recreation (euphoria, excitement, paradoxical excitation) but also that can be potentially dangerous (ataxia, sedation, seizure). These reactions are anticipated with a normal dosing, now you can imagine if you significantly increase the uptake with a very high dose.

If you are a parent, please discuss with your children about this challenge in a calm and posed manner and explain them why it is more dangerous that it is.


Categories
Neurosciences Stroke Uncategorized

[Neurosciences/Stroke] Summary of #ISC20 meeting attendance

A couple of days ago, I attended the second full-day of the American Heart Association International Stroke Conference (#ISC20) that held in Los Angeles, CA from February 19th until February 21st.
Considering this is a meeting mostly driven towards clinical science  (physicians, nurses….), it was a very good year for basic sciences as I had a pretty well loaded schedule of scientific sessions.
My goal is not to provide a detailed description of the findings but really try to keep this summary succint and easy to understand for the layman language. Here are some of the highlights that caught my attention:
* We knew that age is an important predictor of stroke severity and recovery outcome. What we did not knew about is one of these parameters involved is inflammation. A study presented data suggesting that the infusion of young plasma in aged mice undergoing stroke fared better than infusion of old plasma , in terms of infarct size and outcomes. A probable mechanism of action seems to involve a shift of balance in the microglia population (pro-inflammatory vs. anti-inflammatory microglia) and seems to involve exosomes (30-40nm sizes)  diffusion from plasma into the brain.
* Further work on the gut-brain axis and stroke, especially this one on the effect of ischemic stroke on the gut. What is interesting is that an ischemic stroke may have a ripple effect on the gut lining in mice, as it can significantly alter the colon integrity. What was interesting is the sex dimorphism observed, as young female were showing an intact GI lining compared to other group, aligning with the well-established finding that young females fared better than other groups in terms of stroke severity and outcome. Two possible gene candidates identified: MUC4 and CD44, as well as mucin-related genes. Changes in intraepithelial cells (IECs) seems a key factor in such changes.
* Microglia is an important type of immune cells, resident cells inside the brain in resting state. However following injury (DAMPs) or infection (PAMPs), these microglial cells can become activated and trigger the first steps of neuroinflammation. In particular, aging seems to increase the microglial cells population secreting TNF-alpha (a well-known pro-inflammatory factor) suggesting that microglia activation maybe inherently bad for stroke severity and outcome. Therefore, blocking or targeting microglial cell population (by depleting them) would be considered beneficial no? Well turns out maybe not. A study targeted microglia by depleting these cells in both young and old animals using PLX-5622 for 21 days. Interestingly, such treatment resulted in worsened stroke outcome, as the infarct size noted in MCAO group was bigger versus the vehicle group. What was also interesting was such animals showed an increased numbers of monocytes and neutrophils, and an increase in reactive astrocytes in aged animals. There was also changes in the gut microbiota as two genus (Verrucombroia and Akkermansia) were increased in this group, as well as a decrease in Iba1+ gut macrophages.
* Ischemic brain has an effect on the macrophage transcriptome. A study looked at macrophage gene expression profile between circulating monocytes/macrophages in the periphery versus macrophages that migrated into the ischemic injury site. More than 3000 genes were identified as differentially expressed, with an important clustering of genes associated with the peroxisome proliferator-activated receptors (PPARs) including PPAR-delta and PPAR-gamma.
*  MicroRNAs (mIR) play an important role in ischemic stroke injury. In particular a study showed that inhibition of mIR-15a and mIR-16-1 following MCAO (for up to 21 days) resulted in an increased post-stroke angiogenesis (using conditional KO animals). Treatment with AZD0530 resulted in a worsening outcome. A possible mechanism of action may occur via Src-dependent mechanism, that still need some brush-up (is Src activation or inhibition needed?).
* This year, the Thomas Willis award and lecture went to Pr. Maiken Nedergaard (University of Rochester) on the contribution of the glymphatic system. I have to say this is still a very controversial topic that remains highly contested within the BBB field (especially from those interested in fluids movements inside and outside the brain). I have been following as a bystander so I just really keep it on what I have seen presented (and not coming with a knowledge of her papers). According to Pr. Nedergaard, it is a “highly polarized” pumping system, pumping the CSF down to the arterial system, pumped by the glia endfeet processes and clearing in the venous system. According to her, meninges and dura have lymphatic vessels. Amongst things exchanged are ions, lactates and she was also able to observe movements of microspheres up to 1 micron size (aggregates moved to same speed than single ones).
The CSF tracer diffusion was much more present when animals were in sleeping state or under anesthesia, all into the cortex. Similar observations were done in patients as well. The flow is fast and occurs from central to periphery. The CSF accounts about 10% of the brain. According to her findings, tracer was flowing in despite reduced blood flow, all along the cerebral arteries and increase observed mainly in the ipsilateral side.
She also reported a decrease in the volume of CSF after stroke, suggesting that the CSF maybe the source of acute edema. Interestingly, she reported an increase of intracellular calcium influx (known to occur during the ischemic depolarization) preceding the CSF flux, and would still occur later after stroke as the  post-stroke vasoconstrictions would drive such CSF flux.
The rest of the day was marked by some other sessions, meeting old collaborators and friends in the stroke field (an opportunity to meet beyond the emails)  and by the visit of some posters.
Overall, it was a good stroke conference this year, and hopefully next year would allow me to spend more time (it is always tenuous to attend it as it falls right in my busiest semester). Next year? It will take place in the (very) mildly hypoxic mile-high city (Denver, CO), allowing me to drive (or maybe just an hour fly away whichever will be the cheapest option). See you in ISC2021 in Denver!

 

 

Categories
Blood-Brain Barrier Neurosciences Uncategorized

[Sciences/BBB] T Lymphocytes and Cytotoxic Astrocyte Blebs Correlate across Autism Brains (DiStasio et al., Ann Neurol 2019)

I  have been recently approached on social media to discuss about the recent study published by Anderson and colleagues in Annals of Neurology (https://onlinelibrary.wiley.com/doi/abs/10.1002/ana.25610), in which the authors reported the presence of residing CD8 cytotoxic T-cells in the perivascular space of brain samples from ASD patients. I wrote about it, and gave my first impressions about reading the study. After such discussion, I realized that I have an interesting review that worth being shared here on my blog.
Here is the summary of my review of this paper as I wrote it on social media. I made some corrections (mostly spelling and grammar), as well as some changes in the writing style (I wrote these comments on the spur of the moment, as I went through the paper). Also due to copyrights, I will not show any figures and tables from the study.

About the authors: The first author is Dr. Marcello DiStasio (MD/PhD) and the senior author is Dr. Matthew P. Anderson, MD/PhD. He is a clinical faculty of Harvard Medical School and member of the Department of Neurology and Pathology a Beth Israel Deaconess Medical Center, with an affiliation with Boston Children’s Hospital Intellectual and Developmental Disabilities Research Center.  So we have some high profile and experts in neurodevelopment disabilities.

About the journal: The journal is published under Wiley, and is the official journal of the American Neurological Association and the Child Neurological Society. It has an impact factor of 9.49 and is in the top quartiles of neurology and neurosciences journal. Therefore, we have a study published in a very good journal that is relevant to the topic.

About the study design: The study is mostly observational and relies mostly on histological methods (tissue sections followed by staining using chemical dyes and/or antibodies targeting specific proteins). Tissue samples are freshly isolated from postmortem patients (which is a big plus compared to formalin-fixed samples, and opens up the ability to perform protein and RNA analysis if the samples are immediately treated for extraction). The sample size is pretty decent (N=25-30) with a large age spectrum and various types of ASD represented. First interesting to note, 50% of the ASD brain (N=25, not bad) have a history of seizures. Less than 30% of control brains (N=30) have history of seizures. Important thing to consider as a comorbidities and evenutally as a cofounding factor.

About the results: This is my summary of the different figures. By copyright concerns I am not showing the actual figures, but you can overlap my comments to the figures as I have separated them into sections.
Figure 1: Me being cranky, I wanted to see control brain pictures, but not avail. Also with immuno pictures, you have to be super-precautious because there is a high risk of cherry-picking a brain slice, and claim it is representative of all brain. Nevertheless lets discuss it here. On the left panel, we have an H&E staining. Pretty much a vanilla staining. Now, if you want to show a certain protein, you can do it with the right antibody and staining (DAB/peroxidase stain). It appears as the dark brown color. We can see a strong GFAP staining around the vasculature (hollow structure). Astrocytes usually line up blood vessels by forming end-feet process. S100B and ALDH1L1 are pretty standard proteins for astrocytes. However seeing GFAP expression in human astrocytes means these cells are stressed out and are reactive. This is what the quantitative bar graph is telling us. We can also see some CD8 in this perivascular space. These are the cytotoxic T-cells. I wonder what they are doing there, as they can cross the BBB only during brain injury, as microglial cells will äctivate” these endothelial cells and allow white blood cells to adhere on their surface and cross the BBB into a complicated tango dance.
Figure 2: Here there is an attempt of some matrix correlation. GFAP versus CD8 cells. And we can see there is some (expected) correlation between these two (see linear regression and R2) with ASD brains have higher rates of both compared to controls. Interestingly we see similar pattern between ASD that are genetic versus the idiopathic.
Figure 3: digs in more about the immune cells and some correlations (although the scatter of the ASD brains is less convincing here). Overall it seems we have a higher number of lymphocytes in the ASD brain compared to control, both in the white matter (WM, this is where our cables go through) and grey matter (GM, this is where our processing units are) and lepomeningeal (LM, this is our brain surface protective skin, basically the meninges, pours the CSF into the veinous blood). What seems interesting is that at young age we have lymphocytes sitting in our perivascular space, doing nothing(?) and decrease as we age (thats interesting for the non-neuroimmunologist that I am). However, these number at best slightly increase in ASD brain as we age (and I guess not convincingly enough, otherwise the authors would have reported the R2 value), or at least remain the same. With the exception of the medulla (brain stem) most blood vessels show a higher number in ASD brains versus control brains. Interestingly, cortical blood vessels being the predominant population harboring such feature. Very few NK cells to be honest on the panel.
Figure 4: Again this one makes me cringe a bit as a reviewer, because the author do not show the controls data. Yeah, I am pissed. Anyway. Again, perivascular space. Again immune cells highly present (CD3+), CD8 lymphocytes being the predominant type of T cells, in contrast not many CD4 lymphocytes (usually the T helpers, but my immunology is outdated for 20 years at least). Granzyme staining denotes the presence of natural killer (NK) cells. CD20 is a marker for B cells. What is interesting is mostly not much B-cells in either ASD and control brains, the graph more like a refried version of what we already know (higher number of immune cells and CD8 cells).
Figure 5:  is a bit of a useless graph, I don’t see anything that brings us more information than before.
Figure 6: It shows us a Masson trichrome staining. It is a chemical (histological) staining aimed to make collagen fibers visible under a certain color compared to other tissues. Collagens exist in different forms (based on their alpha-fibrils) but the one you expect to see around the vasculature is Collagen Type IV (COL4A1). This one forms a basement membrane (BM) that is like a net around blood vessels. Think about standing on a trampoline. Thats it. Collagen IV forms the net that supports the BBB. Normally, you would expect the BM to be thinned out, this is the case after stroke as cells secrete protein-degrading enzymes (like matrix metalloproteinases MMP2 and MMP9) that will break it down into pieces. But here it is thickened and hhad a bigger thickness than normally. Why so? What does not mean? I dont know. The only time I have seen such things was in transgenic mice overexpression erythropoietin (EPO). These mice had a such high hematocrit that would make blood be like Aunt Jemima corn syrup. If I remember, my former PhD adviser had a collaboration with an electron microscopist and observed similar thickening. Why this is happening is a good question that is likely the next study.
Figure 7: See Figure 5 comment.
Overall thoughts, limitations and outlook: Overall, it is very interesting study, that has some methodological limitations to be noted. First, we are missing any information about the BBB integrity in general, as I wish the authors would have shown some immunofluorescence to compare changes in tight junction (TJ) proteins expression (claudin-5, occludin) in blood vessels and assess differences in TJ strands. The second problem is the lack of information about microglia activation (that would be done by Iba1 staining. The authors noted they performed it, observed a higher expression in ASD brain but decided not to show the data) and more importantly the status of endothelial cell activation (using ICAM1, VCAM1 staining). Are these residing CD8 T cells freshly migrated or just being duck sitting for a while? Is the inflammation status in the brain (and the BBB) ON or OFF at the time of autopsy? What about pro-inflammatory cytokines levels between ASD and control patients? As usual, studies like that opens much more questions that it answers. The second problem is that the study by itself fairly descriptive and observational. It would be interesting how this would compare to clinical findings done in patients. Do we see flares on the MRI indicating of the BBB opening? How does that data compares to patients with MS?
I would also be very careful on making any claim about a “leaky” or “down-regulated” BBB at this point. There is no data about the barrier integrity (TJ complexes integrity) or assessment of the barrier function (for example an MRI scan with gadolinium as contrasting agent) to support the claim. I have seen this claim floating around in non peer-reviewed articles (including in The Scientist), as a BBB expert I would not jump into that conclusion quickly until I see real data on that
This is the type of interesting study, because it opens 100 questions that incentivize to further look down the road. I hope that the authors were able to prepare RNA and protein samples for transcriptome analysis (RNAseq) and proteome analysis (2D-electrophoresis coupled with MALDI) that could help us learn more.

 

 

Categories
Neurosciences Sciences Stem Cells Uncategorized

[Sciences/Neurosciences] Propionic Acid Induces Gliosis and Neuro-inflammation through Modulation of PTEN/AKT Pathway in Autism Spectrum Disorder (Abdelli et al., Sci. Rep. 2019)

Once a wise man said: “Be always wary of scientific studies trumpeted by mainstream news outlets as groundbreaking. Once the smoke settles down, the study in question is rarely groundbreaking, but rather limited with a lot of caveats”. If I have to summarize this study, that would be within the lines of the wise man. Despite what news outlets have been selling, this is a study that has its own merits, but its methodological limitations and caveats outweighs the novelty and significance. Especially considering the publication occurred in Scientific Reports, the response of Nature Publishing Group to the open-access model, this is an extra layer of concern as Scientific Reports prestige and quality has suffered major setbacks in the last few years due to papers retracted for blatant scientific misconducts that should have been spotted by reviewers.

About the authors: We have three authors. The first author seems to be a postdoc, as she apparently graduated from the same program from another lab. The second author maybe an undergrad, although a faculty with the same family name is listed. Finally the senior author is a faculty with an expertise (based on the publication record) on gastrointestinal (GI) tract physiology and pathophysiology. However, none of them seems to have a history of publication in any field of neurosciences. This is an important point, because it explains a lot of methodological flaws that anyone with neural stem cell biology (and brain development) could pick easily.

1. Introduction and hypothesis: The authors are basically using the rationale of changes in metabolomics observed in ASD patients and reported by several studies. In these studies, there are some indications that certain patients on the spectrum (especially those qualified as severely disabled) display an impaired GI function, in particular something we could qualify as something similar to inflammatory bowel diseases. There are studies suggesting that such GI condition is associated in a changes in the gut microbiota, yet with a fairly low resolution (we are able to document changes in a family of bacteria, but not able to pinpoint to the level of Genus species yet). In particular, there is a study that was recently published in Cell Stem Cells (very high impact factor journal) that highlighted changes in mice behavior and gene expression profile of several genes associated to autism following the fecal transplant from patient on the spectrum considered severe (https://www.cell.com/cell/fulltext/S0092-8674(19)30502-1).

In this study, the authors speculate that certain metabolites biosynthesized and/or bio transformed by these class of bacteria are contribution to the symptoms. In particular, the authors consider acetate (AC, CH3-COOH) , propionate (PPA, CH3-CH2-COOH) and butyrate (BA, CH3-CH2-CH2-COOH) as potential culprits, citing studies showing an elevated levels of these small chain fatty acids (SCFA) in fecal cultures of ASD patients compared to control patients. The authors also cite two rare genetic diseases such as neonatal propionic acidemia (PA) and propionyl coA carboxylase (PCC). PA will be very useful to us, because it will help us set what we would consider a pathological level of propionic acid (PPA) in blood. Yet comes the most speculative, and I would say the “jumping the shark” moment of the paper. The authors assume that since processed foods are rich in PPA, such amount of PPA can lead to the development of ASD in the fetal brain during pregnancy. That’s a lot of speculations with little or no layers of evidence. We have here the authors trying to make a statement four to five steps too far from the existing literature for several reasons (and also lakcing the literature backing them up) that be identified as the following:

1.   Where is the literature providing evidence that these SCFA cross the GI tract, at which extent (bioavailability studies)?
2.   What are the levels of PPA (and other SCFA) in the plasma/serum levels of neurotypic patients versus patients on the spectrum? For patients suffering from PA, I have found an old study referring to serum PPA as high as 0.337-1.35mM, with a normal level about 0.00337mM (https://www.jpeds.com/article/S0022-3476(81)80004-2/pdf)
3.   How much of PPA can cross the blood-brain barrier? This is an important question to answer. We can try to build on the analogy of the SCFA to ketone bodies (acetoacetate, beta-hydroxybutyrate) that are formed when someone is fasting or forced into a ketogenic diet. Considering that patients suffering from GLUT1 Deficiency Syndrome are showing improvement when put on a ketogenic diet (with BHB levels around 2-3mM), we can speculate these compounds can cross the BBB readily.
4.   How much of PPA can cross the placental barrier? I don’t have any clue either.

These talking points are important because it will determine if the experimental design of a study is sound or deeply flawed, which eventually will set the quality of the paper and the robustness of the conclusion made. That’s something I should mention by now, is that both the authors and the news outlets have been very fond of superlatives and trying to sell that paper at much higher level that it is meant to. Not only it is scientifically inadequate to make extraordinary conclusions without highly robust data to support them, but also it is nowadays dangerous to do so as such studies will be used by scammers, charlatans and other snakeoil salesmen to promote their supplements claimed to “cure autism” and use these type of studies to claim their products is supported by science.

2. Materials and methods: The authors used neural stem cells (NSCs) derived from fetal tissues (obtained from Life Technologies/Thermofisher) and maintained in a classical medium formulation aimed to maintain these NSCs in their pluripotency stage. The cells were passaged no more than three times, according to the author. This is important, as NSCs/NPCs passaging over time will coax them towards the astrocytes lineage compared to neurons (in terms of development, neurons appear earlier and mature earlier than astrocytes).
Now there is something intriguing. The authors claimed they looked at PPA and BA at concentrations ranging from 0.1mM (that would be physiological), 0.5, 1 and 2mM concentration. Considering such treatment would reproduce the fetal brain, we have to factor in what is the amount capable to cross the three barriers: GI barrier of the mother, the placental barrier and finally the BBB. However, the authors never showed what happened to these concentrations except the 2mM which is limit deadly (remember? This is the one that is detected in newborns having the rare genetic mutation). Are the authors trying to model the effect of PPA to model such diseases or are they assuming that the amount of PPA in processed found will be high enough to put the pregnant women into a severe metabolic acidosis? I don’t know but that like a red flag.
The differentiation of these NSCs into neurons/astrocytes were left to occur in a fairly random fashion, as the authors use the same medium used for NSCs maintenance but without growth factors. I think this is an important issue here as we may have a significant variability in terms of yield between passages in particular when it comes to neurons/astrocytes ratio (personal communication with Clive Svendsen).
Otherwise, nothing else really fancy and classical techniques found in any neurosciences studies: Immunocytochemistry, neurite outgrowth, qPCR…

3. Results:
3.1. Figure 1: I am a bit perplexed from what I see and from what I get when it comes to quantifications. The first issue I have is the lack of scale bar. A scale bar tells you how many pixels equals to a length. For example, a 512×512 pixel image may indicate you that 100 pixels equals to 50 micrometers. Here you have to trust that the experimenter did not fudge the data, crop the pictures and really show you a 10x magnification.
For simplicity, I will focus on the Day 10. My concern is about the health of the neurospheres in some of the groups, in particular the BHB-treated group. You can see in controls; we have nice rosette-shaped neurospheres with a dark core reflecting a dense. In contrast, look at the BHB treated group. These neurospheres are small, frail and lack the morphology observed in control. I wonder if BHB at this concentration is showing signs of toxicity? If yes, the authors were not concerned at all by this issue. And this is something concerning. If BHB is neurotoxic, so how can we make a conclusion to BHB as inhibitor. There are ways to show the viability of these neurospheres: Hoechst staining, propidium iodide staining, Fluorojade staining……..Because of this important issue, I will not consider the BHB treatment as valid.

Slide1

If you compare the data shown in Fig.1B and 1C compared to the quantification made in Fig.1A (and shown in the bottom), you can see we have a certain discrepancy here. I would skip the issue in the y-axis labelling (the correct symbol for micrometers is µ (mu) not n(nu)), but compare the 10 days timepoint to the data we actually obtain from the Fig.1A. In scientific publications, you have to be sure that what your representative blot/micrograph picture shows matches your quantitative analysis. In other words, what I see in the micrograph pictures in Fig.1A should be reflected in Fig.1B and 1C.
Then explain me why the differences in diameter reported is not as different between my quantitation (using ImageJ internal functions) is different from the one displayed. How do the authors justify the use of SEM instead of SD, except for making the graph look nicer (you can see the data actually suggest a much more variability that likely undermine the statistical difference)? How does the authors explain the 3x difference in neurosphere counts between me and them? Did they crop the pictures? If so they should have accounted for, and highlights the importance of having a scale bar in micrographs pictures and normalizing such data to a surface area (e.g. pixels2, µm2, mm2….)

3.2. Figure2: These are immunofluorescence pictures of plated neurospheres allowed to differentiate by their own on Matrigel-coated plates (Cultrex). The pictures are okay, although not very convincing for some and certainly not suitable for quantification. The use of flow cytometer is definitively a go-to when it comes to assessing cell populations.

Slide2

We are also here having a mixed results and missing important cell markers. First, the authors should have performed a nestin staining, as nestin is one of the markers present in NSCs/NPCs. Second, the use of GFAP as astrocytes marker has to be taken with a big grain of salt. I am not sure experts in the field would have let this fly with just one marker. GFAP can be expressed by NSCs and NPCs. Showing at least two markers per cell lineages (NeuN/bIII tubulin for neurons, S100B/GLAST1 for astrocytes) would have been much more convincing. bIII tubulin antibody (in particular the one used in this paper) is known to have a very strong non-specific staining. A good bIII tubulin would show nice neurites. I have attached a picture of an iPSC line developed by Sigma-Aldrich. You can use it to compare so you can see what a good bIII tubulin and a good GFAP staining should look like in NPC-derived astrocytes. Here we have just some blobs (that indicates a possible non-specific immunoreactivity) that dangerously overlap with GFAP. Technically, you cannot have a neuron that express both GFAP or bIII tubulin. It is either or but not both. R&D Systems has a nice interactive map that shows you the different cell markers expressed by the neural lineage as its differentiate into neurons and astrocytes here: https://www.rndsystems.com/pathways/neural-stem-cell-differentiation-pathways-lineage-specific-markers

What I am supposed to do with that?

3.3. Figure 3: The authors looked at both GFAP and bIII tubulin at mRNA levels (PCR) and protein levels (by ELISA). I would have personally put the PCR data first, followed by the ELISA data. The PCR data was normalized to GAPDH and the DeltaDeltaCt method was used, which is good, the authors also have represented the apparent bIII tubulin or GFAP/GAPDH ratio, which is good.  However, I am more skeptical on the ELISA data. The reason why? The data is represented as micrograms of protein/microliters of cell extract. I am skeptical why the authors did not run a Western-blot analysis for these two housekeeping genes, since you expect a lot of proteins being expressed. The authors also forgot to mention if they have diluted the samples or just added the crude extract at is. This is important because you can easily blunt the accuracy of your ELISA. LSBio is honestly a cheapskate when it comes to showing a standard curve, unlike more established ELISA kits manufacturer such as R&D Systems or Abcam, that will show you their standard curves and tell you the coefficient of variation in them. The maximum concentration of the standard curve is 1000pg/mL or 1ng/mL, with a detection range of 15.63-1000pg/mL and a sensitivity of 9.38pg/mL. The authors reported concentrations for GFAP was 0.8-3 pg/ml according to their graphs. Something is wrong here, and we have at least 2 reviewers that completely miss that. Are the authors telling me that they were able to detect GFAP and bIII tubulin below the sensitivity level (9.38 and 313pg/mL respectively)? Give me a break! I would also have advised the authors to normalize their concentrations into something meaningful like mg of proteins. It is easy to take a fraction of the cell lysate and measure the total protein concentration by BCA. I ask my students whenever they use an ELISA for quantifying a cellular protein to normalize their amount detected (pg/mL) to a total protein concentration (mg/mL) which allows us to normalize the data. Failure to do this normalization is like showing a Western-blot without a proper loading control (e.g. actin, GAPDH….).

3.4. Figure 4: In this figure, the authors are trying to show the expression of GPR41 (aka free fatty acid receptor 3 or FFAR3) in those cells. Honestly, this is my breaking point of tolerance. First, the authors underwent some cherry-picking of the data, showing you only the PPA treatment in astrocytes (where is the BA treatment? Where are the BHB treatments?) and only the BA treatment in neurons (where is PPA? Where are BHB?).
I am also very skeptical that what the authors call astrocytes are really astrocytes looking. What we see in Fig.4A looks very similar to 4B: very thin cytoplasmic projections looking like neurites. Only neurons form neurites in cultures. Astrocytes have more a flat-shaped feature, sometimes a bit fusiform like shape. Again, the GPR41 protein expression is really up when you have tons of PPA given (mM and more). How come this went through peer-review unabated and have at least 2 reviewers did not notice this gross conundrum in the data?

4. Rest of the figures and conclusion: I can go further with this paper. It was looking very interesting and promising, but the lack of expertise from the authors quickly percolated into loose and inconclusive data. This is the kind of paper you wish the authors would seek feedback from across the street, from some faculty with a neuroscience background and give them an honest feedback to make this paper good and scientifically sound. What we have indeed is a half-baked study, served as the next big thing since sliced bread. Not only the data is far from convincing of the claims made my authors (I would probably accept as a possible model for modelling PA or PCC, but this paper IS ABSOLUTELY NOT SHOWING THAT PPA IS CAUSING AUTISM for several reasons below:

1.   It does not account for the PPA levels found in normal persons, even less provide a study showing PPA levels in people eating processed foods (if such dietary habit even lead to such outcome).
2.   It does not consider that in order to be valid the authors have to show that you have a 100% bioavailability of PPA across the GI barrier, the placental barrier and the BBB, which are not reported or cited by the authors in any credible form.
3.   It does not account that the levels used as so ridiculously high that a pregnant mother would deal with a possibly deadly metabolic acidosis.
4.   It also ignored that BHB was showing signs of neurotoxicity.
5.   There is a worrisome pattern of data cherry-picking, with groups popping in and out intermittently, sometimes even in a complacent manner. This is a no-no and an unacceptable behavior that has no place in any respected peer-reviewed journals. Why did the reviewers overlooked that issue?
6.   There are several inconsistencies in the data, especially whether the axis labels are botched or if the authors really provided measurements that were nornally impossible to reach (below detection limit).

This paper should at least had a “major revision” to fill the gaps. Yet, it went through at least 2 reviewers and none of them were able to see the obvious methodological flaws. As a reviewer for Sci Rep on a seldom basis, I am very concerned about the quality of review provided by the journal in the recent years, especially in light of series of retraction. Conclusions? The news outlets have been trying to sell an overhyped paper that does not live much under scrutiny. This is just “same old, same old” when it comes to journalistic reporting on science (trying to fudge it as groundbreaking), but also opens a dangerous precedent. I will bet that within 12 months, there will be some quack doctors and snake oilsalesmen that will claim they can cure autism by selling you supplements aimed to reduce the PPA or by selling you a dietary fad book, claiming it will cure your child autism by dietary restriction. I guess the keto diet will soon join the casein-free/gluten-free diet as outdated and have another fad being served as dietary torture to children on the spectrum.

Categories
Neurosciences Sciences Stroke Uncategorized

[Sciences] Restoration of brain circulation and cellular functions hours post-mortem (Vrselja et al., Nature 2019)

You may have heard about that groundbreaking story last on “pig brains being revived” sounding almost like a scenario of a zombie movie. Let’s say science journalism love to use superlatives and sensationalistic headlines to grab few more clicks and views.
As usual, my skepticism was to first look at the paper and see how the claims hold on. The publication behind that “pig zombie paper” is the study from Vrselja and colleagues published in Nature last week and available here: https://www.nature.com/articles/s41586-019-1099-1
So what is about this story? First it is published in Nature, a top-tier peer-review journal. Second it is a huge paper, coming from Yale University. The paper was initially submitted on February 22, 2018 and got accepted March 1st. You can say a bit more than a year and that suggest that this paper likely went at least two rounds of review and probably more than three peer-reviews (three were named as well as other anonymous). One of the peer-reviewer was Pr. Constantino Iadeccola (Cornell University, NY), a “rock star” in the field of cerebral blood flow (which nicely match for the paper).
Overall, it is a very good paper with some reservations on the greater impact that I will explain later.
To understand the paper, you need to understand first that as until now we consider the brain highly dependent on continuous cerebral perfusion with blood flow to survive. The brain is highly dependent on oxygen and glucose (at least 20% of our daily uptake is taken by this tissue that only represent less than 2% of the human body weight).
We assume that if you stop flowing the brain with blood (e.g. cardiac arrest), you will die within minutes from massive and irreversible brain damage. The whole idea of this paper was: “what if we could maintain a blood flow for 24 hours, can we maintain some neurological function?”. In particular, the authors have developed a kind of artificial blood, cell-free, called BEx. It contains a hemoglobin carrier called Hemopure(R), glucose/pyruvate, as well as a cocktail of neuroprotective agents, antibiotics and some echogenic agents (to measure blood flow). The caveat is that as a control the authors used a simple saline solution without glucose and pyruvate (see supplementary tables below). Considering the importance of glucose for the brain tissue, and the absence of glycogen storage in that tissue, I would argue that this is a non-negligible flaw in the experimental design, giving a serious advantage to the BEx and maybe even overestimating the BEx activity.
Screen Shot 2019-04-17 at 2.06.26 PM

Nethertheless, let’s continue the discussion. Pig brains were not obtained from pigs euthanized for the sole purpose of the experiments, but rather obtained as waste from the slaughterhouse. Thats ethically much more acceptable, even 3R-friendly (as it valorize animal tissues considered as waste) and much more easy for obtaining an IACUC approval. About 300 post-mortem brains were used, I guess mostly for the development and optimization of the technique. The sample size (N) appears to be 32 pigs/group, which is very good for statistical power of analysis.
The surgical procedure (to connect these brain to the system) was about 10 minutes of warm anoxia, which would probably represent a severe cardiac arrest in which CPR is not performed immediately. They exposed these brain to either 1 hour or 10 hours post-mortem interval (PMI) without flow, with control perfusate or with BEx. Note that the perfusion to occur happened about 3 hours since the initial brain flush, the surgical preparation appears indeed tidious, but reproduce a pulsate blood flow similar to what would happen in animals. They also cooled down the brain to 25ºC, which is known that cooler temperature improve the chance of reducing brain damage (the common sense is that drowning in an hypothermic environment (frozen lake) increase your chance of resuscitation compared to drawing in a normothermic environment (swimming pool). The experiment lasted 10 hours for all groups, except the 1 hour PMI group.
The first results shown demonstrated the presence of a functional flow inside the brain tissue, and some vascular reactivity, using nimodipine infusion (a Ca channel blocker commonly used to reduce blood flow) and showing changes in blood flows. In other words, there is a proof of principle that it works.
The second result looked at changes in cerebral edema as a crude estimation of the blood-brain barrier (BBB) function. The control perfusate showed an increased water content, which is not surprising as some of our in-house data (and other studies) suggesting that the BBB function has a greater dependence to glucose than to oxygen when it comes to maintaining the barrier integrity. Since the CP is glucose-free, that is not surprising. The BEx group of course fare better (same level than 1 hour PMI group) but wonder how it would have fared if the CP contained the same amount of glucose and pyruvate. My personal thought is why did not the authors performed a gadolinium imaging of these two brains? They provide some T1 scans, which are nice but confirming changes in the BBB leakiness using gadolinium as contrasting agent would have been better.
Figure 3 show us a series of tissue staining of these brain, in particular from the hippocampal region. As expected, the Nissl staining worsened in the PMI, the presence of CP partially improved the situation and the BEx was similar to the 1 hour PMI and has the lowest cell death (as imaged by active caspase-3). When you look between CP and BEx, the difference is not that dramatic and makes me wonder that if we had the right CP formulation (with same glucose/lactate content), we would unlikely have a difference between CP and BEx, suggesting that perfusion with a saline solution oxygenated and with the correct amount of glucose can do as well than a more complex one.
Figure 4 show that the perfusion with BEx help to maintain astrocytes and microglial cells alive and functional (as measured by the secretion of pro-inflammatory cytokines following treatment with LPS).
Figure 5 show that there are some functional neurons present in BEx, capable to show electrophysiological activity. Small activity (not enough to be detected by EEG) but measurable by patch-clamp analysis.
Overall, it is a nice paper, considering it got published in Nature. There are some interesting stuff, but there are also some questionable limitations and caveats that I would have pointed as a reviewer and expected reviewers from Nature to have pointed it before letting it accepted. It shows that no matter what, never blindly trust a paper even if published in Nature.
The idea is very interesting and can help us better understand the post-mortem brain. It also raises the importance of CPR or any procedure aimed to keep a steady flow in the brain after injury or cardiac arrest and maybe worth considering it.

Categories
Blood-Brain Barrier Neurodegenerative diseases Neurosciences Stem Cells

[Sciences/BBB] X-linked adrenoleukodystrophy and blood-brain barrier: latest insights from preclinical and clinical studies

Two studies came out of interests in the last few weeks about the dysfunction of the blood-brain barrier in X-linked adrenoleukodystrophy (ALD): an in vitro study from Azarin and colleagues (I know Dr. Azarin as we collaborated on several publications together) using patient derived stem cells to model the blood-brain barrier in a dish (https://www.ncbi.nlm.nih.gov/pubmed/29615068) and a brief communication from clinical data showing an improved blood-brain barrier function (by MRI) in ALD patients treated with stem cells (https://www.ncbi.nlm.nih.gov/pubmed/30635285).

Before I go into details, I think it is important to discuss about ALD. ALD is a X-linked genetic disease (it affects mostly boys, as they carry only one copy of the X chromosome) affecting one in 18’000 patients and with a grim prognosis. It usually onset between 4-10 years old children, resulting in their rapid neurological deterioration and eventual death within 2-5 years. As of today, there is no treatment.

ALD pathophysiology is mainly occurring as a demyelinating disease. Myelin is a complex fatty acid acting as an insulant around nerves axons, by ensheathing such axons very similarly as a plastic insulant around copper wires in electrical wires.  In ALD patients, there is an abnormal processing of very long chains fatty acid (VLFCA), resulting in its buildup in the serum and in the brain white matter, resulting in abnormal immune response in the brain and neuroinflammation. About 50% of ALD patients will display a childhood cerebral ALD (ccALD), as displaying the clinical features mentioned. Because it is primarily a disease affecting neurons, a lot of research have been focusing on neurons and ignored non-neuronal cells.

These two studies add to the list of neurological diseases associated with ALD. First, the study by Azarin and colleagues (University of Minnesota-Twin Cities) show that brain endothelial cells (BMECs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALD patients have a deficit phenotype compared to controls, have poorer barrier tightness and maturation. Moreover, they show that such BMECs have lipid inclusions (a cellular feature of lysosomal storage disorders) and express genes involved in inflammation at higher levels than controls. Interestingly, they also identified a polymer (PEO-PPO) can alleviate the dysfunction and partially restore the barrier function. It was interesting to see this study finally out, as I have seen the poster of it at the recent Gordon Research Conference.

Second, the study by Lund and colleagues (University of Minnesota-Twin Cities too) noted an improvement of the BBB (using MRI imaging and gadolinium as contrasting agent) in patients that received an hematopoietic stem cell transplant (bone-marrow transplant) as seen by a reduction in Gd leak by day 30 and improvement of patients outcomes. At this point, there is no clear evidence of how it works (the author speculate that some monocytes from the donor may migrate in the brain and attenuate the neuroinflammation) and if there are an overall improvement of the patient function.

Still, these two articles are interesting as it bring an insight of the contribution of the BBB in the ALD pathophysiology and further demonstrate the importance of the BBB as a contributing factor in several neurological diseases.

Categories
Blood-Brain Barrier Junk Sciences Junk Sciences Neurosciences Sciences Uncategorized

[Sciences/Junk Sciences] Zeolites, blood-brain barrier and “Autism Detox” scam.

Recently, it came to my attention of another scam popped up on social media. This scam came in form of the “Autism Detox” page on Facebook coming with the following description:
IMG_2005

“Zeolite”, “blood-brain barrier”, ‘detoxify toxins and heavy metals” and “cellular level”.
Incredible how much amount of BS claims can be packed in such a small vaporizer. Not only this was enough BS, the owner of this page went the extra mile and claims it is an “autism detox” as well.
I call this an utter amount of BS and since I am a scientist, I will explain why it is an utter amount of BS.

1. What are zeolites?
Zeolites are crystalline structure made of aluminum, silicium and oxygen. These crystals are formed by the aggregation of 4 oxygen atoms around aluminum Al3+ and Silicium Si4+ (notice how Avers that yells “shark” on aluminum in vaccines are fine absorbing aluminum from zeolites).
These frameworks of AlO4 and SiO4 can form 3-D geometrical structures harboring charges and possibly acting as a caging structure as shown below (Moshoeshoe et al., Am J Mat Sci 2017):

As you can see different structures exist. Now, which zeolites are used in the product described in this “detox”? According to the vendor website (https://www.coseva.com/toxin-removal/advanced-trs/), clinoptilolite (CLI) (amongst water and a proprietary formula). According to Mosheoshoe and colleagues, CLI harbors the following chemical composition ((Na,K)6(Si30Al6O72) •20H2O)) and harbor the following crystalline structure:
Clinoptilolite

Notably, CLI also display one of the lowest cation exchange capacity (CEC) of 2-2.6 mEq/gram. In summary, CLI is a small zeolite crystalline structure with limited cation exchange (against Ca2+, K+ and Na+). First, it shows that these compounds have a molecular weight exceeding the size recommended for small molecules (~832 Da>500 Da), a ring size bigger than the tight junction pore (5.6 Angstroms>4 Angstroms) and an non-negligeable amount of molecular charges. All these features make CLI very unlikely to cross the blood-brain barrier and no studies have provided a direct experimental evidence that CLI crosses the BBB.

2. Does zeolites even cross the GI tract?

Good question! The only paper that I found discussing about zeolites is a paper from Cefali and colleagues (Cefali et al., Pharm Res 1995). Unfortunately I cannot access the paper but the abstract provides two important parameters: Cmax and AUC. In particularly, it also provides the value of aluminum hydroxide (yep, that stuff found in vaccines).
Cmax is indicative of the maximal concentration reached upon administration via extravascular route (IM, PO or SC). The AUC is representative of the total amount that reached the circulation from the time of administration until the time the drug becomes undetectable in blood. From the abstract we have the following information The mean plasma silicon AUC values (+/- S.D.) were 9.5 +/- 4.5 [Note: Zeolite A], 7.7 +/- 1.6, 8.8 +/- 3.0, 6.1 +/- 1.9 mg.hr/L [Note: Aluminum Hydroxide] and the mean plasma silicon Cmax values (+/- S.D.) were 1.07 +/- 1.06 [Note: Zeolite A], 0.67 +/- 0.27, 0.75 +/- 0.31, 0.44 +/- 0.17 mg/L [Note: Aluminum Hydroxide] for Zeolite A, sodium aluminosilicate, magnesium trisilicate, and aluminum hydroxide respectively. Although mean silicon AUC and Cmax values were elevated when compared to baseline after administration of the silicon containing compounds, only the AUC from Zeolite A reached statistical significance (p = 0.041). The mean plasma silicon Tmax values (+/- S.D.) were 7.9 +/- 6.4, 5.8 +/- 4.6, 6.9 +/- 6.3 and 8.5 +/- 3.4 hrs for Zeolite A, sodium aluminosilicate, magnesium trisilicate and aluminum Hydroxide respectively.”. Since we have a Cmax and AUC value for Zeolite A and aluminum hydroxide very similar, we can assume that both compounds may likely show similar bioavailability. Considering the bioavailability of Al is very low (0.3%), it is very likely that zeolite and CLI may not show a higher value that this one. Thus, out of 100g ingested of zeolite, maybe less than 0.3g will likely reach the bloodstream. In conclusion the amount of zeolite capable to cross the GI is very small and considering the volume of a TRS “Detox” (28mL), the amount of zeolite capable to cross the GI tract after swallowing a whole bottle of it is likely to be ZERO.

3. What about the rest of the claims?
As far we have seen:
1) CLI absorption at the GI tract is likely close to ZERO, even if you sip a whole bottle at once (see 2).
2) CLI cannot cross the BBB because of the physicochemical constrains (see 1). The only paper listed in Pubmed is a letter written to a journal with no scientific evidence or experimental data backing up the claim (https://www.ncbi.nlm.nih.gov/pubmed/23224491).
3) The claim of detox is utterly BS: there are two organs that do it for you. The liver and the kidneys. Thats it.
4) Heavy metal detox mostly occurs via renal (kidney) filtration. Even if zeolites can trap ions like Na+ or K+, I still have to find a paper that shows me it can trap heavy metals (Cd2+, Pb2+, Hg2+…..). CLI has been shown to only trap three ions (Ca2+, Na+ and K+) with the poorest ability.
5) Claiming that autism be cured is not fallacious but criminal. Until now, there is no cure for autism. There is no evidence that chelating ions cure autism (chelation therapies have even been proven to be dangerous and responsible for the death of at least one boy). There is also no published mechanism of action demonstrating how a treatment can reverse a condition mostly identified as genetic.

 

Categories
Blood-Brain Barrier Neurosciences Sciences Uncategorized

[Neurosciences/BBB] Brain Endothelial Erythrophagocytosis and Hemoglobin Transmigration Across Brain Endothelium: Implications for Pathogenesis of Cerebral Microbleeds (Chang et al., Frontiers Cell Neurosci 2018)

I usually don’t post BBB papers on my blog because most of the time they address concepts or answers questions that are not relevant for the public in general, but I thought this one was an interesting paper to share. This is an original article published by Rudy Chang and colleagues in Frontiers in Cellular Neuroscience las month. It is open-access, that allows everyone to access to it. Another interesting feature is the disclosure of the reviewers that help improve the peer-review process and transparency.
Why I found this paper interesting? Its because it propose a novel mechanism of cerebral microbleeds, without affecting the tight junction complexes. In the field, when we consider brain bleeds, we consider a loss of the barrier function and a massive brain leakage. Such phenomenon occurs when you have an hemorrhagic stroke due to an aneurysm, or due to a arterioveinous malformation resulting in an unstable blood vessel. The presence of blood in the brain parenchyma is harmful for two reasons:
1) You are injecting a volume inside a closed space (cranium) that will lead to an increased mechanical pressure (intracerebral pressure) and ultimately brain damage by tissue crunching.
2) Red blood cells (RBCs) may be damaged (hemolysis) and release their content into the extracellular space. Heme is toxic at high concentration (through mechanisms that yet to be identified) and can further damage neurons via generations of free radicals and other damage-inducing signaling pathways.
In this article, they demonstrate that you may achieve a similar outcome than brain bleeds without having a leaky BBB. In particular, this study demonstrate the ability of damaged RBCs to cross the BBB via transcytosis (via an engulfment inside the BBB and the exit to the other side). For this study, they used bEND.3 cells (an immortalized mouse brain endothelial cells) and compared mouse RBC that were considered healthy or induced damage via tert-butylhydroperoxide (t-BHP). t-BHP induces oxidative stress (via the release of radical oxygen species such as hydroxyl radicals), in this case leading to the exposure of a particular phospholipid named “phosphatidylserine” (PS) from the inside to the outside of the cell surface membrane.
In this study, they demonstrated that oxidative stress mattered in RBC cell adhesion to b.End3 cells. Treatment of b.End3 cells with t-BHP or LPS (a bacterial membrane lipid, commonly used to induce an inflammation state at the BBB) failed to yield similar results. in addition to demonstrating the ability of RBC to adhere on b.End3 cell surface (the first step needed for cellular transcytosis), they also demonstrated the ability to have these cells to engulf and get trapped into these cells. This process appeared slow and it took about 18-24h to see a significant number of RBCs inside the cells. Finally, they show that such RBCs were capable to migrate through the b.End3 monolayers and popped out in the other side. Similar outcome was observed in vivo, but to a certain extent.
It is a very interesting study, because it can maybe explain some aspect of diseases associated with RBCs such as cerebral malaria (we can imagine that Plasmodium may use RBCs as a Trojan horse to cross the BBB). However, I also have some criticism of the study. First, it uses the b.End3 that has fairly poor barrier function (TEER<100Ohms.cm2), much less than the tightness expected in vivo (>2000Ohms.cm2). The second issue is inherent to working with non-human cells. Do we have the same outcome when it comes to the human BBB and human RBCs? Maybe this phenomenon is exclusive to rodents and may have limited impact in humans. Finally, and as seen with the in vivo data, RBCs maybe able to cross the BBB but they maybe likely get retained by 1) the basement membrane supporting brain endothelial cells and 2) rest in a limbo state in the perivascular space (a virtual space between the basement membrane and an external protein mesh called “glia limitans” wrapped around cerebral blood vessels). We have some hints as RBCs appear juxtaposed near the vasculature, as stuck by a mesh surrounding the vessels. I don’t think that RBCs can cleave such mesh because I assume they don’t have the molecular machetes (matrix metalloproteinases) to cut their way through. Yet, I think it is a very interesting paper, that work to be investigated with a human BBB model, using a more robust model.

Categories
Drug Delivery Neurosciences Sciences Uncategorized

[Sciences/BBB/Drug Delivery] Red blood cell-hitchhiking boosts delivery of nanocarriers to chosen organs by orders of magnitude (Brenner et al., Nat Comm 2018)

I know, I know I have been fairly quiet. I have to tell that between attending a scientific meeting, teaching a summer course, taking care of two grants proposals and finally handling three manuscripts. However, sometimes I also like to share some papers published in the field that are interesting or bringing novel ideas or concepts. This one has been suggested by one of the author and this is his quick summary:
We bind nanoparticles to the surface of red blood cells. These nanoparticles are going to be released from the red cells once they pass the first capillary bed. Therefore we can concentrate the nanoparticles in the target tissue. In addition, these nanoparticles potentially can carry one or multiple selected drug(s). So if we inject intravenously the particles are going to be release in the lungs but if we inject through the brain arteries the nanoparticles will be release into the brain vasculature.
It is a very nice of work here published in Nature Communication (I consider this is one of the top OA journal to get published in) and you can download the full-text here.
You can also appreciate this paper likely went into at least one round of review, with a time period of about 7 months between it got accepted for review and accepted for publications.

In this paper, the authors have been working on trying to develop alternative drug delivery carrier, in that case use red blood cells as “piggyback” cells to enhance drug delivery. They tried different formulations on isolated RBCs and identified some suitable for carrying antibodies or proteins.  They called these piggybacking RBCs as RBC-Hitchiking (RH) (I wonder if it is some Easter egg towards the “Hitchiking Guide to Galaxy”).

Upon identification of the right nanoparticle materials, the authors investigated the distribution and the delivery of the conjugates in different organs, as liver and lungs. What is interesting is the amount of injected dose recovered is much higher in the RH than the free-circulating one, in particular in lungs, whether they are bare-naked or bound with a protein. These nano-carriers can be delivered to endothelial cells.

But the interesting snack-bite from this paper is the intra-arterial injection in the carotid artery, in which there was a significant increase in nano carriers delivery in the brain.  Nano-carriers alone show a %ID lesser than 1% (that is about what you expect from delivering antibodies from an IV route towards the brain)  to over 10%.   The delivery was also maintaining the ipsilateral injection site, which is good considering you are likely to treat one brain hemisphere.

Now, time for me to be picky and kind of wish we had information on the PK profile, especially if this approach increased the stability of the nanocarriers. It would be also interesting to see how this technique fare in a experimental disease model (for instance a xenograft brain tumor and see if you can deliver targeted chemotherapy in mice).

Nevertheless it is a good paper that take us out from the classical nanoparticles formulation and try here an innovative and novel approach in drug delivery.

 

Categories
Junk Sciences Neurosciences Uncategorized

[Vaccines/Junk Sciences] “Murine hypothalamic destruction with vascular cell apoptosis subsequent to combined administration of human papilloma virus vaccine and pertussis toxin” (Aratani et al., Sci Rep 2016 – RETRACTED) Lesson from a paper that went completely fritz on the scientific method

You maybe heard about the recent retraction notice of a study published in Scientific Reports that raised concern about the safety of Gardasil(R) (HPV vaccine) and got retracted this week. Another anti-vaccine paper that bit the dust. I would have say, I am not surprised at all. Anti-vaccine studies have this very annoying habit of either being a proven fraud (remember the latest Shaw paper?), botching the experimental protocol with omission of proper controls (thats the Exley paper I have reviewed) or conveniently sweeping under the rug some data that are not fitting the narrative (that goes for one recent paper published by Gherardi).
But this one is interesting at several levels, because there is a bit of blood-brain barrier in  it, and also adds to the list of papers retraction in Scientific Reports and recent threats by scientists in the editorial board to resign from their positions due to ambiguous and unjustified decision on a flawed paper (Disclaimer: I have a study authored in this journal and I have peer-reviewed for them a couple of times).

To be honest, I only heard about this paper during the last weekend and took some times to read the paper. I will be honest, I don’t see any scientific fraud in the sense of data manipulation. What concerns me is how such a botched study could even pass through peer-review process? Considering I self-impose a quality of standard in my manuscripts and still get challenged by peer-reviewers, seeing such junk studies getting a free pass is a bit vexing. I agree that open-access may not have the same stringency in terms of peer-review filter but considering Scientific Reports as part of Nature Publishing Group, you expect a rigor found for any Nature-related journals applied in this journal too.

But lets go through the paper, it is retracted but you can still access it here.

What is the wrong with this paper?

1. The experimental design in terms of groups

The first problem arises from Figure 1.

Aratani_Fig1

We have six groups: vehicle (PBS), pertussis toxin (PTX), Gardasil(R) (MSD, HPV vaccine 4-strains) (G), G+PTX, EAE and EAE+PTX. Let’s breakdown first these oddities.  Why the author has included a PTX group, even more adding PTX with Gardasil? There is not much explanation in the text explaining the rationale (also the writing style is odd, very odd. I completely understand that the first author is not a Native English speaker. As an ESL myself, I completely understand that). Also, I am not aware about a higher risk on contracting pertussis upon vaccines.
The second aspect is the use of the EAE mouse model. EAE stands for experimental autoimmune encephalitis. It is the “gold standard” for a mouse model of multiple sclerosis (MS). The idea behind is to inject a brain protein (myelin basic protein or MBP), which will trigger an immune reaction (as the brain is a immune-privileged organ) and result in symptoms similar to MS. I would understand to compare the incidence of Gardasil(R) on MS patients by comparing EAE mice versus non-EAE mice but that is never the case (they even administer PTX to a sub-group).
So here we already start with a wrong experimental design: it just make no sense. A more rationale approach would have been the following:
Vehicle (PBS), Gardasil (G), EAE, EAE+G. That would have saved two groups and precious lives sacrificed in another useless study.

2. The experimental design in terms of statistics and power of analysis

Another important issue is the blatant dismissal of consideration of biostatistics and the power of analysis in the experimental design. For those that are not familiar with scientific research, you have to ensure you have a statistical meaning to your data to ensure the effect observed is real and not due to simple coincidence. This statement is especially true when working with vertebrates animals. Any animal experiment has to be approved by the institutional IACUC that ensure you have a clear idea of what are the purpose of your experiments, how you will ensure a humane treatment to animals and also have an optimal number of animals to achieve a statistical significance.
An important aspect for in vivo (animal) studies is to achieve at least a sample size of 8 or more animals per group (n=8). From Figure 1a, we have already a violation of this as only the G and the G+PTX have enough animals (n=14 and 21 respectively). All other groups are below the n=8 threshold. In addition, you have very different number of animals per groups (control has n=6, EAE has n=5) making the statistical power weak and also restricting the use of common statistical methods such as the use of ANOVA (ANOVA recommends that all groups have an equal number of samples).

3. The experimental procedure and treatment

This is where the firestorm came in: the experimental data. So lets bring this to the table: “Groups of 11 week-old female C57BL/6 mice were intramuscularly administrated 100 μ l of Gardasil or phosphate-buffered saline (PBS) for a total of five times. Ptx was intraperitoneally administrated 2 and 24 hours after immunization. The Gardasil vaccine or Ptx were administrated at 2- weeks or 4-week intervals“.
A key element in a paper is the methods section and this one utterly failed. Based on this information I have absolutely no idea why they injected five times (Gardasil immunization is maximum 3 times), why they injected the PTX right after the immunization (2 and 24 hours, suggesting a double-induction) and when they injected the Gardasil and PTX (how do they separate the 2-weeks versus the 4-weeks? When did they started?).
Also the use of 11 week-old female is not reflective of a human case scenario. If we approximate 1 human year to about 3.6 mice-days, you would expect to use young mice (males and females, to have a gender-balanced study) that are about 6-weeks old (~43.2 days). That would be about 12 years, the age of puberty.
Here we are basically injecting HPV to adult females, which is known to not provide an additional benefit as such population may have already been exposed to HPVs.
The next problem is the injection dose. It is 100microL, thats the equivalent of 0.1mL. A single dose of HPV is 0.5mL. Lets ignore the scale law and assume a mouse is an equivalent to a human. A 50th percentile weight at age 12 is about 40kgs. Lets assume a 6-weeks old mice to be about 20g.
If we assume 0.1mL injection to a 20g mouse, then the human dose-equivalent would be about 200 dose-equivalent injected at once! This is a serious issue because there is absolutely no chance that such things to happen in a lifetime (at grand maximum you may have 3-4 HPV injections, spaced in time). Also, if we assume the age scale, we are expecting to have mice receive their two doses within 48 to 96 hours, not 2 to 4-weeks.
I am not even entering the rationale to inject PTX right after immunization, which is utterly no-sense and just scramble defining the effect of HPV versus the effect of PTX. Another disastrous example of how this paper was flawed from the beginning.

4. Failure to report weight and clinical score

If we want to follow an EAE protocol, it is important to show the evolution of animal weight over a period of times (up to 15-21 days) as well as a clinical score. The clinical score is a well-described protocol in which features found in EAE mice are score from mild (tail flaccid) to severe (inability to move hindlimb or complete immobility).
These two graphs are almost present in any EAE paper outside in the literature.
I assume this is what Figure 1b and 1c wanted to show but very poorly. Indeed Figure 1c does not really show up anything. We dont know when these data have been taken, we have no idea about the onset time of symptoms and furthermore we have no indication of a statistical differences. This is already a waste of data.

5. The constant cherry-picking of the data and incomplete picture

The methods used are honestly laughable: some hematoxylin-eosin staining (a common histological stain that does not tell much unless you have massive brain damage or the growth brain tumor),  Kluver-Barrera staining (for myelin staining), TUNEL stringing (for apoptosis), a behavioral test relegated in Supplementary Figure S1 (in which the author thinks that a P-value of 0.1 has a statistical meaning).

Aratani_Fig2

Aratani_FigS1
Where is the Evans blue extravasation staining to show a disrupted BBB? Where are the GFAP staining to show astrocytes activation? Where are the CD11b and F4/80 staining to show microglial cells activation and macrophages infiltration? Nowhere to be seen. We have to comptent ourselves with some miserable histological staining in Figure 2 and 3. Also no-one of the data about EAE is never shown past Figure 1. Figure 4 is even more laughable as the author only shows the staining of the G+PTX, giving the middle finger to the reader to how such staining looks like in vehicle, or G.

Aratani_Fig3

How can the author be confident that it was the Gardasil treatment, not the PTX treatment (despite being mentioned as a BBB disrupting toxin) being the sole contributor of all this?

6. Conclusions

Another anti-vaccine study, another case of botched science resulting in a junk paper, the sacrifice of animals over a useless experiment. That should not have been passing through the peer-review filter at all because of its deficiencies, yet was able to go through. If I was the reviewer behind it, I would have been ashamed to have this paper not outright rejected for major flaws in the study. Should we assume that the author recommended some complacent reviewers  to this paper? Or should we question the integrity of the editorial board in accepting papers for publication that fail to address some scientific integrity? Again, anti-vaccine studies shows that they cannot challenge vaccine safety and can only make fool of themselves by producing junk studies like this time.

The first and senior authors of this paper produced a paper that is so bad, they should feel ashamed to even had published at first.