Schematic of how COVID-19, through activation of the complement system, may damage the endothelial cells of the blood-brain barrier, leading to microglial activation, astrogliosis, and neuronal death.

Scientists discover how COVID-induced brain “fog” (i.e., brain damage) may occur

So this new study comes from the journal Brain, and it fills in many gaps in the current understanding of how COVID-induced “brain fog” occurs. The research team was led by Dr. Avindra Nath, clinical director at National Institute of Neurological Disorders and Stroke (NINDS).

For some time, scientists have speculated that the virus, which has been found in the parenchyma (i.e., tissue) of the brain, may enter through one or more of several routes. A direct route would be through the olfactory bulb and related nerve in the nose. Another would be via the vagus nerve. Alternatively, the virus could infect endothelial cells and thereby gain access to neurons and their supporting cells known as glia. Lastly, the virus could enter through a disrupted blood-brain barrier (BBB), along with other cells such as leukocytes (aka white blood cells).

It’s important to note that any combination of these may be taking place; or some other, not yet described pathway may be involved.

Blood-brain barrier. Endothelial cells form tight junctions (TJs) around the capillary to maintain integrity. Pericytes surround TJs, and the pericytes in turn are surrounded by the foot processes (also known as pseudopods) of astrocytes, which maintain and nourish the vascular structure. As depicted, those same astrocytes support and interact with neurons as well. Microglia are sentinels, monitoring the area for foreign substances, pathogens, injury or other items that might cause alarm.

The researchers found specifically that the process of neurological damage in COVID implicates at least one of these pathways: that of a disrupted BBB. Yet they also demonstrate that in this cascade glia become activated as well, which further damage various areas in the brain.

The blood-brain barrier is designed to shield the brain against toxins or pathogens circulating in the blood that could cause brain infections if they were to permeate the barrier. Yet the BBB also allows vital nutrients to pass through. Its other function is to help maintain relatively constant levels of hormones, nutrients and water in a state of homeostasis. Maintaining the brain’s delicate balance is critical.

An article at ScienceAlert describing the research explains:

The scientists discovered that antibodies produced against COVID-19 mistakenly targeted cells that form the “blood-brain barrier” – a structure designed to keep harmful invaders out of the brain while allowing necessary substances to pass.

​Damage to these cells can cause leakage of proteins, bleeding and clots, which elevates the risk of stroke.

​The leaks also trigger immune cells called macrophages to rush to the site to repair damage, causing inflammation.

Immunostaining of proteins involved in breaching the blood-brain barrier due to COVID-19. C1q and C4d are involved in the complement system; IgG and IgM are immunoglobulins.
Complement activation and immune complexes. Immunostaining for (A and B) C1q, (C and D) C4d, (E and F) IgG, and (G and H) IgM shows minimal staining in the brains of non-COVID-19 controls and extensive deposition on endothelial cells in the blood vessels of COVID-19 patients.

The proteins discovered by the team were immunoglobulin A (IgA) and immunoglobulin M (IgM). According to Wikipedia, IgM “is the first antibody to appear in the response to initial exposure to an antigen.” The M in IgM stands for macro, and it references the size of the particle. It is much larger than other proteins in its class. Thus, to find that it has crossed the BBB in these patients means that the BBB indeed suffers serious disruption in this cascade.

The process

News-Medical Alert also helps describe the process occurring here:

Collectively, multiple large proteins, like immunoglobulin M (IgM), that ordinarily do not pass the blood-brain barrier were present in the perivascular areas of the deceased COVID-19 patients, which showed the deterioration of vascular integrity in those patients. High fibrinogen levels were seen in the vicinity of the blood vessels, with concentrations gradually declining as the distance from the vasculature increased, indicating a porous blood-brain barrier.

Imagine a crime scene where a burglar has tracked footprints throughout the house, pointing to a route of escape. As the trail continues away from the scene, the material on the bottom of the shoes leaves fewer and fainter clues. This is what is happening with regards to the BBB: the evidence shows that the trail of fibrinogen diminishes with distance. The damage is preserved in the parenchyma.

Fibrinogen, a glycoprotein produced in the liver, helps form blood clots by occluding blood vessels. It is normally not detected in brain tissue, except in cases of pathology such as thrombosis or a leaky BBB.

Fibrinogen interacting with PrPc, a protein in the brain. PrPc is a precursor to PrPsc, which is prion-like.
Fibrinogen (in red).

In some regions of COVID-19 patients, neurons and glial cells stained for fibrinogen, presumably representing uptake from the parenchyma,” according to the study. This means these cells absorbed the protein into their cell bodies.

Another protein that assists in clot formation is Von Willibrand factor (VWf), which is involved in platelet adhesion and aggregation. (VWf’s effect on platelets is included in the schematic that heads the diary.) Note that, according to Wikipedia, “[e]ndothelial cells are attached to the subendothelial collagen by VWf”; these same cells are a primary target of SARS-CoV-2.

Per the study, “Platelet aggregates were present in all brain regions of the COVID-19 patients,” as well as meningeal and choroid plexus blood vessels. The regions examined via immunohistological staining were “the temporal and frontal lobes, olfactory bulb, basal ganglia, thalamus, hippocampus, midbrain, cerebellum, pons, and medulla oblongata,” according to News-Medical Alert.

According to a press release by NINDS,

“Activation of the endothelial cells brings platelets that stick to the blood vessel walls, causing clots to form and leakage to occur. At the same time the tight junctions between the endothelial cells get disrupted causing them to leak,” Dr. Nath explained. “Once leakage occurs, immune cells such as macrophages may come to repair the damage, setting up inflammation. This, in turn, causes damage to neurons.”

When I first saw the description regarding macrophages, I thought that perhaps this meant microglia were activated, as microglia are the resident macrophages of the brain. However, the original study clearly delineates that the macrophages in question are those from the periphery of the body, leukocytes—specifically monocytes—that morph into macrophages once they are activated by the complement system. (For reference, a monocyte is the largest type of white blood cell; a macrophage is a cell that eats pathogens and other cellular debris.)

Immunological complement pathway cascade.
A simplified schematic of the complement system cascade.

The complement system is involved in the body’s immune response. Per Britannica.com, it is comprised of more than 30 proteins “that act in concert to help eliminate infectious microorganisms.” Normally quiescent in blood, when they receive a signal from the immune system “[t]he signal sets off a chemical chain reaction in which one activated complement protein triggers the activation of the next complement protein in the sequence.” 

From the study:

To determine whether there was activation of the complement cascade, we immunostained for several complement molecules. Complement components 1q and 4d (C1q, C4d) were present on endothelial cells and platelets. This was associated with deposition of IgG and IgM.  

“Deposition of immune complexes was seen in all regions of the brain,” reported the study. Endothelial cells showed the presence of “C1q, C4d and complement component complex (C5b-9) along with IgG and IgM.” Additionally, “C1q, IgG and IgM were also present in the perivascular regions,” most often in what is known as the extracellular matrix (where neurotransmitters and other such material are found) but also “in some glial cells and neurons. Focal areas of C1q deposits were also seen on the myelin sheaths of axons.”

Graph of extent of neuronophagia in the brains of COVID-19 patients.
(G) The number of foci of neuronophagia was significantly increased in the brains of COVID-19 patients compared to non-COVID-19 controls (****P H) There were significantly more foci of neuronophagia in the hindbrain compared to the forebrain of the COVID-19 patients (**P = 0.0071).

As the schematic that heads the diary shows, the monocytes transform into macrophages once they pass the BBB. Once there, they begin to devour what they determine to be pathogens and debris. But also this cascade activates the brain’s microglia as well. Now both types of macrophages are rampaging through the brain. All regions of the brain. 

The activated microglia begin to perform what is called neuronophagia. It is as terrible as it sounds. It is bad enough that in the world we might encounter creatures known as brain-eating amoebae; in this instance, though, the danger is due to our own immune systems.

The NINDS data reinforces similar findings about COVID-19 and neuronophagia reported in 2021. Said one of those researchers at the time, We know the microglia activity will lead to loss of neurons, and that loss is permanent.”

Immunostaining of microglial damage in the brains of COVID-19 patients.
Microglial nodules and neuronal injury. (CF) Clusters of CD68+ microglia surrounding neurons were present in the (C) hippocampal CA1 region of Case 2, (D) thalamus of Case 8, (E) pons of Case 7 and (F) solitary nucleus of the medulla.

The macrophages tended to affect the hindbrain more than the forebrain. The forebrain includes the cerebrum, basal ganglia and thalamus. The hindbrain contains the pons, medulla and cerebellum. In fact, the study specifically pointed to what are known as Bergmann glia, which are astrocytes that surround and protect Purkinje cells of the cerebellum, the most tightly, densely packed area of the entire brain.

The implications

According to the press release,

Had the patients in the study survived, the researchers believe they would likely have developed Long COVID. “It is quite possible that this same immune response persists in Long COVID patients resulting in neuronal injury,” said Dr. Nath.

This study adds to the body of research, especially of that published this year, that spells out what COVID-19 might do, long-term, to the brain. We know that the brain undergoes a significant loss of gray matter volume (in many of the same brain regions cited by the current study). We know that, even in mild disease, microbleeds driven by thrombi occur throughout the body, including the vascular areas of the brain. We know that COVID-19 ages the brain an equivalent of up to 20 years of ageing. And we know that the disease can shave anywhere from 7 to 10 points from a person’s IQ.

Other things to keep in mind: COVID has been shown to damage oligodendrocytes, which are glial cells that wrap axons in myelin, providing insulation for the propagation of electrical impulses. Additionally, as astrocytes become reactive, they stop regulating the extracellular space between neurons, including junctions between synapses; this means that glutamate, a neurotransmitter, may accumulate in such junctions until they become excitotoxic to cells, causing them to undergo apoptosis, a process of programmed cell death. The disrupted BBB indicates such an initiation of astrocytic reactivity, at least among those that maintain the BBB via foot processes.

These factors, plus the reactivity of microglia, demonstrate profound dysfunction in the brain as a whole.

Speculations

As previously mentioned, there was a COVID diary I penned where I described the research of a team that had discovered that cells in the body undergo a process called pyroptosis. I used a metaphor in the title, and someone in the comments framed that as a misrepresentation of the science.

In a way, though, the cells do attempt a self-sacrifice, an immolation of sorts, in order to try to save the host as a whole. Yet, as I intimated at the time, I think the cells are misguided, if such can be said. The cells in question are monocytes, and they spectacularly burst themselves open with chemical alarms in order to signal other cells to ready themselves for the COVID battle.

But monocytes are the same ones that wander across the BBB.

As I said in the previous diary:

I personally am concerned about what is going on in the brains of these patients. We have reason to believe that the blood-brain barrier is disrupted or disturbed in COVID, which may make it easier for these monocytes to find their way into the brain parenchyma, where the neurons and supporting cells reside. Microglia are the brain’s equivalent of monocytes/macrophages. We already know that microglia are implicated in profound neuroinflammation in COVID patients; we just don’t have (as far as I know) definitive proof that pyroptosis is occurring there. I have my suspicions.

We still don’t know if pyroptosis is occurring beyond the BBB, once the cells squeeze themselves through the disjointed endothelial cells in a process called diapedesis. But I think this study by NINDS has shown brighter light on the possibility that this may be so. We know now, due to this study, that monocytes indeed are traveling into the parenchyma.

Figurative brain disease or destruction.

Other studies, not dealing with SARS-CoV-2 in particular, show that fibrinogen, when it disrupts and disperses past the BBB, is one of the first signals of neurodegeneration. This is known to be true in the case of Alzheimer’s disease. Coupled with that is the fact that scientists now see COVID as initiating a disease process much like Alzheimer’s. More than that, it has now been shown that SARS-CoV-2 creates amyloid proteins in the brain. Lastly, COVID upregulates tau, a prion-like particle that, when the hyperphosphorlated version proliferates, leads ultimately to the host’s demise.

Will COVID create its own type of dementia? If so, I have already speculated that it would resemble corticobasal dementia (CBD), based on the findings of one particular study (conducted on non-human primates) that showed a concentration of viral effects in the basal ganglia. CBD manifests several parkinsonian features and also several of CTE (chronic traumatic encephalopathy).

Multifocal loss of neuronal processes due to breach of blood-brain barrier in COVID-19 patients.
Microglial nodules and neuronal injury. (B) Double labelling of CD68 (brown) and calbindin (red) in the cerebellum of Case 9 shows multifocal loss of neuronal processes.

The NINDS study specifically found that the hindbrain saw more damage than the forebrain. That the cerebellum seems to incur the greater portion, at least in this limited study (nine postmortem patients, ten controls), may indicate that the type of dementia that COVID might induce will be more localized and might manifest in a manner different from CBD. Striking the midbrain and other structures lower in the brainstem, the virus may create a disorder that more resembles ALS or some other dyskinesia.

My ultimate speculation is that this disease hits the pons or somewhere thereabouts more than anywhere else, perhaps right in the vicinity where the vagus nerve meets; I’ve held this view privately for some time now, but I feel more comfortable speculating about it out loud now, seeing that the NINDS study showed that the choroid plexus is an area of involvement. The choroid plexus is where the BBB coincides in proximity with the cerebrospinal fluid, and such a juncture (the third ventricle) happens to be very near where the pons and the cerebellum meet.

But I make that speculation very tentatively! I’m still learning several underlying, rather foundational facts about gross anatomy, neurochemistry, microbiology and the like. But I would like to think I’m on the right track. I welcome feedback from those with the relevant background.

I also wonder whether the cerebellum sees more damage due to the Bergmann astrocytes. These glial cells are especially permeable to calcium influx. As previously noted, excessive glutamate in the extracellular space can cause excitotoxicity; this process directly involves increases in Ca2+ (calcium ions) as well as upregulation of NMDA receptors, which enhances glutamate sensitivity. All of this would help drive cell toxicity. There may be synergy here between Purkinje cells and their associated astrocytes in terms of this toxicity.

Brain capillary endothelial cells showing virus particles within cytoplasmic vacuoles.
A, Brain capillary endothelial cells showing virus particles within cytoplasmic vacuoles (← arrow) B, Blebbing of viral particles coming in/out of the endothelial cell wall (circles) The relationship of virus particles (arrows←) to the endothelial cells (virus ingress/egress) is depicted. Note the dense inner core and densely stained periphery of viral particles. C, Endothelial neural cell interface showing a cytoplasmic vacuole filled with viral particles in various stages of bud formation (arrow←) adjacent to the basement membrane within the neural cell (frontal lobe). D, Neural intracytoplasmic vesicle showing viral-like particles. Insert: Detail on viral particle exhibiting electron dense centers with distinct stalk-like peplomeric projections. Scale bars are shown at the bottom left/right of each figure. BM, basement membrane; EC, endothelial cells; IV, intracytoplasmic vesicles; NT, neural tissue; RBC, red blood cell

One thing I would like to add before closing this diary. The researchers of the NINDS study assert that “SARS-CoV-2 has rarely been found in the CSF of patients with CNS symptoms, and autopsy studies have either failed to find the virus or the virus has been found in the brain at only low copy numbers without associated inflammation, which cannot explain the widespread pathology.” I found this odd, as I had read elsewhere that the virus had been found in parenchyma. I compared the sources given by both studies and found that the NINDS study lacked overlap with the other by one source. And that source indeed shows that the virus is taken up by endothelial cells of the capillaries of the brain. (See image right.)

This discrepancy does not go toward uncovering why the researchers at NINDS found no virus particles in the brain tissue itself. The authors themselves wonder if the host’s own antibodies attach to the ACE2 receptors of endothelial cells, which would kick off the cytokine storm cascade. It’s unclear. Though, considering the study of monocytic pyroptosis, I am tempted to ponder if that is the path by which cytokine storm starts in earnest. Either way, it is unresolved as to why it seems that the virus possibly is neurotropic yet across studies is not regularly found in parenchyma.

Takeaways of the study

As summarized by News-Medical Alert:

  • The scientists assessed nine COVID-19 patients who died during the initial COVID-19 wave between March and July 2020.
  • Multiple large proteins that ordinarily do not pass the blood-brain barrier were present in the perivascular areas of COVID-19 patients.
  • This showed deterioration of vascular integrity.
  • High fibrinogen levels were seen in the vicinity of the blood vessels, with concentrations gradually declining as the distance from the vasculature increased, indicating a porous blood-brain barrier.
  • The vascular injury indicators were more frequent across the hindbrain.
  • The endothelial cells harbored high platelet endothelial cell adhesion molecule 1 (PECAM-1) titers; however, the cause of heightened PECAM-1 is unknown.
  • The PECAM-1 serves as an adherent for platelets and their aggregates attached to the endothelial cells. The stimulated platelets occasionally led to the obstruction of the tiny blood vessels.
  • The tissue factor (TF) and CD74 genes help to distinguish between COVID-19 patients from controls and aid in the thrombi development.
  • Relative to controls, the COVID-19 patients had higher levels of the signaling pathways for semaphore and the ras homologous guanosine diphosphate-dissociation inhibitor (RhoGDI), which correlates with vascular permeability. These inferences accounted for microinfarcts.
  • The team discovered 25 angiogenesis-associated genes contrastively expressed in the COVID-19 patients’ brains. They also identified dysregulation of the signaling pathways implicated in angiogenesis.
  • Endothelial cells and platelet aggregates possessed IgM and IgG masses co-localized with many complement cascade components.
  • The existence of C4d, C1q, and C5b-9 were indications of an activated classical complement network. The team also discovered C1q and C3 deposition in macrophages and endothelial cells.
  • Immunohistochemistry revealed cellular infiltrates of macrophages, CD8+, and CD4+ T cells in COVID-19 patients, in line with previous reports.
  • Despite the low amount of T cells, CD8+ cells exceeded CD4+ cells, and B cells were relatively rare.
  • Neurons and glia absorbed serum proteins like complement and fibrinogen.
  • Astrocytosis was most common in the perivascular domains.
  • The cerebellum and other hindbrain areas experienced a multifocal depletion of neurons.
  • Sirtuin and other genes and signaling routes connected to oxidative stress and deoxyribonucleic acid (DNA) impairment were increased in COVID-19. Most pathological observations were more frequent in the hindbrain.
  • The authors suggested that antibody-triggered cytotoxicity targeting the endothelial cells probably led to neuroinflammation, vascular leakage, platelet aggregation, and neuronal damage.

ONE LAST THING!

Get vaccinated! Not only has it been shown to lessen the severity of COVID in breakthrough cases, the pyroptosis study showed that vaccination almost completely stifled the reaction of monocytes in blood plasma. (This is important if those monocytes also somehow traverse the BBB.) Lastly, an observational study just reported that two or three vaccine doses may cut the risk of contracting Long Covid. Every reason exists for getting vaccinated and staying up on your schedule thereafter.


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