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Using a three-dimensional model of engineered human tissue, researchers replicated the pathological effects of the blood-brain barrier under ischemic conditions and used drugs to prevent or lessen the damage. The lab model will be used to test new therapies, their potential neurotoxicity, and make it easier to understand how the blood brain barrier works, experts say.

Using a three-dimensional model of engineered human tissue with six different cell types, researchers at Wake Forest School of Medicine replicated the pathological effects of the blood-brain barrier (BBB) under ischemic conditions and used drugs to prevent or reduce the damage.

The spheroids have all the qualities of human BBB functionwith expression of tight junctions, adherens junctions, adherens junction-associated proteins, and cell-specific markers. They also have the same proportion of human microvascular endothelial cells, neurons, astrocytes, microglia, pericytes, and oligodendrocytes.

The lab model will be used to test new drugs in the pipeline and their potential for neurotoxicity. The model will also make it easier to understand how the BBB works to protect the brain and to model human brain diseases, experts told Neurology Today.

We developed a much-needed model of an entirely natural BBB and show that we can reverse many of the problems that occur in acute stroke, said senior author Anthony J. Atala, MD, director of the Wake Forest Institute for Regenerative Medicine.

In our three-dimensional model, every cell contributes. The development of tissue-engineered three-dimensional brain tissue equivalents can help advance the science toward better treatments and improve patients' lives. The study was published online on June 17 in Nature Scientific Reports.

Much of the day-to-day work on these studies was carried out by the paper's first author, Goodwell Nzou, PhD, who created the model during his doctoral studies at Wake Forest.

To study the model, the scientists decided to test what goes wrong with the BBB during stroke. They depleted the organoids of oxygen and observed an inflammatory cascade set in motion to stop the damaging chain of events that occurs during stroke.

By knowing at the cellular level what is going on, we can start thinking about therapies to stop this process, explained Dr. Atala.

Not surprisingly, the more oxygen removed from the cellular environment, the more damage there wasincluding an increase in proinflammatory and oxidative stress molecules. The investigators looked closely at the neuroinflammatory cascade and observed that the length of exposure, not just the amount of oxygen reduction, seemed to play a role.

The cytokines that were released disrupted the integrity of the BBB, and over time the cells began to lose their capacity to function normally. The scientists were able to measure the chemicals during the cytokine storm: chemokines and cytokines, heat shock proteins, transport proteins, tight junctions, and basement membrane protein expression.

Next, they used anti-inflammatory agentssecoisolariciresinol diglucoside and 2-arachidonoyl glycerolto reduce the effects of inflammation and the other pathological processes underway.

The treatment was effective at ameliorating the damage and improved tight junction distribution, said Dr. Atala.

The investigators also tested a free radical scavenger and anti-inflammatory endocannabinoid that have been shown to model inflammation and cell death. Again, it worked to alleviate the response to the hypoxic environment.

Dr. Atala, a regenerative medicine specialist, believes that this disruption of the BBB may be used as a crack in the door to get targeted treatments through. When we used these agents we were able to manage the response and observed positive effects, said Dr. Atala. By using this model that replicates in many ways the human physiological response to hypoxia we can now look at agents that can impact things in a positive way.

The critical role played by the BBB in maintaining homeostasis within the central nervous system makes it clear why BBB breakdown is evident in many neurological disorders, they wrote in the paper. During ischemic stroke, in particular BBB breakdown leads to edema and hemorrhage.

These pathologic consequences worsen secondary brain injury and significantly contribute to cognitive impairment. Hence pathological effects of hypoxia on BBB function may be critical in understanding strategic therapeutic targets for BBB maintenance and recovery during and after neurologic injury, the scientists wrote.

The scientists are now designing studies of other pathological conditions, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Dr. Atala said that neuroimaging studies have shown BBB breakdown and inflammatory processes underway in many neurodegenerative conditions.

The development of three-dimensional organoid models of the neurovascular unit offers a unique opportunity to study the effectiveness of novel therapeutics for neurological diseases in a cell-based system that takes into account intercellular communication, said Patrick Ronaldson, PhD, an associate professor of pharmacology, physiological sciences, and neuroscience at the University of Arizona College of Medicine.

Intercellular communication between endothelial cells, glial cells, mural cells, and neurons is a property that is often lacking in many of the in vitro model systems that are routinely used for CNS drug discovery and development.

Dr. Ronaldson added that three-dimensional neurovascular unit organoid models can also enable the identification of novel compounds that can protect the vasculature from further injury. Such utility may facilitate critical breakthroughs in treatment of neurological diseases where blood-brain barrier dysfunction and neurovascular injury are known pathological characteristics, such as ischemic stroke, traumatic brain injury, and Alzheimer's disease.

Despite this advance, however, he said there are many unanswered questions regarding the application of this three dimensional organoid model to drug discovery and development. These questions specifically relate to the magnitude of blood-brain barrier leak observed in the organoids and the ability to study drug transport at the neurovascular unit in a multi-transporter environment.

This study evaluated BBB permeability using only large molecular weight tracers such as immunoglobulins, albumin, and fluorescently-labeled dextrans, he explained.

This is a missed opportunity because permeability to smaller molecules could still persist. Such a leak of small molecules can occur even if large molecular permeability is restricted and can contribute to exacerbation of neurovascular pathology or neurotoxicity. Understanding whether therapeutics can protect against small molecule paracellular permeability is critical in the advancement of these compounds to the clinic.

Additionally, the researchers only evaluated organoid expression of one therapeutically relevant transporter (that is, P-glycoprotein, also known as MDR1), Dr. Ronaldson said.

There are many other transporters such as breast cancer resistance protein and organic anion transporting polypeptides that are critical determinants of drug disposition at the neurovascular unit, he continued. For this three-dimensional organoid model to be truly applicable to neurological drug discovery and development, it is important to evaluate the expression and function of the many other transporters that are known to be clinically relevant.

Costantino Iadecola, MD, the Anne Parrish Titzell professor of neurology and director and chair of the Feil Family Brain and Mind Research Institute at Weill Cornell Medicine, said: The findings are anticipated based on previous stroke studies: the impact of hypoxia on BBB constituents, cytokine production, oxidative stress, etc., and the protective effects of free radical scavengers, anti-inflammatory cannabinoids. All these events and protective approaches were already known and validated in animal models but failed in humans.

There is little mechanistic novelty and therapeutic advance [with this paper], he said, but the positive aspect is that the model could be used for higher throughput screens for new drugs.

But, Dr. Iadecola added, one caveat with the current study is that only a particular segment of the cerebral vasculature was examined (capillaries and related cells) and that only hypoxia was tested. This is not the same as ischemia produced by an arterial occlusion in vivo, which is associated with intravascular clotting, distal microembolism, infiltration of inflammatory cells, no-reflow phenomenon, and more, he said.

Drs. Atala, Ronaldson, and Iadecola had no relevant disclosures.

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A Model of the Human Blood-Brain Barrier Shows the Effects... : Neurology Today - LWW Journals