Final Report: Dr Paul Holloway – Developing human mini-brain circuits to reveal new insights into stroke

New technologies now allow researchers to culture (grow) and study human neurons in a dish. Typically, these cell cultures are a random entanglement of cells, unlike the ordered connections of the brain.  

Dr Paul Holloway has harnessed microfluidic technology, creating a device within which, individual nerve cells can form organised circuits that mimic the human brain. This human-focused ‘mini-brain’ enables stroke conditions to be re-created and is the first of its kind, advancing research into stroke injury. 

Replacing animals in stroke research: Using human cells and mimicking stroke conditions 

Strokes happen when blood flow to the brain is disrupted, depriving nerve cells of oxygen and glucose, causing them to die. This nerve damage can spread to connected areas of the brain but how this happens isn’t fully understood. Animals are typically used in stroke research but the stroke needs to be artificially induced by cutting a blood vessel in the brain. This is followed by investigations into ways of stopping this injury from spreading.  

In this research, Dr Holloway has used human induced pluripotent stem cells (iPSCs). These are cells derived from human skin or blood, which have had a ‘factory reset’, so can be reprogrammed back into an embryonic-like state. This enables the development of an unlimited source of any type of human cell needed, including nerve cells, thereby replacing the use of animals with more a human relevant model. 

Dr Holloway has shown how oxygen and glucose levels can be controlled and lowered in specific compartments of the microfluidic model, mimicking stroke conditions. This simulated stroke-like environment means he can study how nerve cell injury caused by oxygen and glucose starvation, can spread through a network of otherwise healthy nerve cells. This is the first lab-based technique that allows researchers to study the impact of various oxygen levels on nerve cell circuits, something that was previously only possible in animals’.  

Mimicking the stroke environment

To create stroke-like low oxygen conditions (known as ‘hypoxia’), Dr Holloway used chemicals to ‘mop up’ oxygen, optimal channel dimensions to control microfluidic flow and oxygen permeable materials within the device. He and his team investigated the effectiveness of a number of chemicals in depleting oxygen to find the most effective one, called Pyrogallol.

The hypoxic conditions also enabled them to induce some of the changes seen in important proteins. Under normal oxygen conditions, a protein called HIF is routinely broken down but during hypoxia, the protein stays around, and has a key role in controlling hundreds of genes that affect the survival of connected nerve cells. Dr Holloway used specialised microscopes to show that there were high levels of the HIF protein in the low oxygen compartments, further evidence of the successful creation of a stroke-like environment.

Image: A microfluidic device filled with coloured dye. Two channels used to grow nerve cells (red/pink) are connected by microchannels (akin to corridors). These guide the growth of nerve cells which extend their ‘axons’, long branches extending from the nerve cell, connecting to other nerve cell axons. The oxygen scavenger (in green), created by mixing chemicals (yellow and blue,) runs alongside the chambers where the nerve cells grow and remove oxygen to mimic the conditions during a stroke.

Validating the ‘mini-brain’ tool 

A key part of the project has involved optimising the imaging of the nerve cells. Dr Holloway honed the images to ensure they were as clear as possible, in preparation for the team’s continued development of this, and other animal free models.  

Illustration of nerve cells connected in an orderly arrangement across microfluidic channels within the ‘mini-brain’

Another important step was to show that the nerves in the ‘mini-brain’ were connected and working as they should. Dr Holloway used a chemical to kill the nerve cells on one side of a connecting channel (highlighted in orange in the image). The chemical used, effectively ‘over-stimulated’ the nerve cells, causing them to die, a phenomenon seen in strokes.  

When this chemical was blocked, Dr Holloway saw the nerve cells connecting to each other in an ordered way and working as they should. When the chemical wasn’t blocked however, microscope images showed fragmented, dead and non-functional nerve cells. 

Collaborating and sharing of the research 

Dr Holloway is using the results of this research as the basis for an application for wider funding, in order to study specific ways in which nerve cell injury may spread following a stroke, and to further demonstrate the power of animal replacement technologies. 

He’s also preparing to publish these results in an international peer reviewed Journal later this year. This will raise awareness of this technique and how it can help to answer specific questions about what happens during a stroke, questions which could previously only be answered within the complex nervous systems in living animals.

Dr Holloway is attending the Microphysiological Systems World Summit this year. This is a collaborative conference where the latest scientific achievements in human-relevant models and tests are presented and advances and challenges are discussed to help accelerate and implement developments in this field 

Paul presented his work, which included some of the work that is funded by Animal Free Research UK, to his international collaborators on a visit to Harvard University in April 2023to encourage collaboration within the network and get some of the network groups to use some of the animal free methods he has been developing. Many of the groups are quite interested in using these animal- free models. 

Masters student from the University of Manchester will be undertaking a year-long placement, using the techniques developed in this work, to investigate how human circadian rhythms influence nerve cell death and gain insights into potential new treatment avenues. 

Dr Holloway was recently awarded (Oct 22) the Einstein Center 3R visiting Expert award in Berlin where renowned scientists from around the world foster in-depth scientific exchange and international collaborations. He gave a lecture that featured some of the work done in this research. Visiting Experts – EC3R  

The team are now also utilising this technique as part of an international collaboration exploring the role of circadian rhythms (the internal process that regulates our sleep–wake cycle), as there could be differences in strokes that happen in the day, compared with at night. Most stroke research involves using nocturnal rodents who are inactive during the day when the research is done but are poor representatives of human strokes as the majority of these happen when we are active, in the daytime.  

Dr Holloway has also been interviewed and shared his research on the Animal Free Research Labcast (a free Podcast).