Haemorrhages in foetal brain tissue associated with the presence of SARS-CoV-2: How SARS-CoV-2 impacted our PhD bringing new and important findings.

In October 2020, we started to observe haemorrhages in the human fetal brain tissue that we received from the HDBR. We were previously using the tissue from HDBR to investigate features of typical cortical development, but we quickly realised that we would need to change track and investigate why these tissue samples contained these haemorrhages, and if this was linked to the COVID-19 pandemic we were in the middle of.

Two and a half years after receiving the first haemorrhagic sample arrived in the lab, we published the paper in Brain (https://doi.org/10.1093/brain/awac372). Here, we (first author Marco Massimo and second author Carlotta Barelli) share the story behind the paper. 

How did you get started on this project?  

Marco Massimo: I always say that this project ‘happened’ to us, as it was totally unexpected. In our lab we work with human fetal brain tissue provided by the Human Developmental Biology Resource HDBR (https://www.hdbr.org). It was October 2020 when HDBR sent us the first haemorrhagic sample. Although haemorrhages in fetal cortex have been observed, they are extremely rare, and initially we were all surprised to see such an injured sample. Over the following months we kept receiving more and more samples presenting haemorrhages and it got to the point that we couldn’t do our experiments investigating typical cortex development anymore! As you all will remember, in October 2020, in the UK, we were in the middle of the SARS-CoV-2 pandemic. Covid cases per day were remarkably high and a vaccine was yet to be offered to the public. We had never received this kind of injured samples before the pandemic, and it is highly unlikely to observe such a high number of haemorrhagic samples in such a short amount of time. That’s why we thought that the highly unusual number of haemorrhagic samples might have something to do with the ongoing Covid19 pandemic. So, this is how we began this project, we hypothesised that those haemorrhages could be associated with SARS-CoV-2, and, with the support of HDBR, together with our collaborators in Trieste and Edinburgh, we started investigating this option. 

Carlotta Barelli: I had already been working in the lab for a couple of months as an undergraduate internship student when we started receiving an abnormally high number of human fetal brain tissue samples displaying cortical haemorrhages. These samples could not be used to study normal brain development and in December 2020 we had received enough of these haemorrhagic samples to start analysing them. Due to the timing, we began investigating whether SARS-CoV-2 could be involved in the injuries observed in these fetal brain tissue samples.

Can you summarise your findings? 

MM and CB:  In our study we report SARS-CoV-2 infection in human fetal brain in association with haemorrhage, disrupted endothelial integrity and infiltration of immune cells in the developing cortex. 

Cortical haemorrhages were linked to a reduction in blood vessel integrity and an increase in immune cell infiltration into the foetal brain. Our findings indicate that SARS-CoV-2 infection may affect the foetal brain during early gestation and highlight the need for further study of its impact on subsequent neurological development. 

Video abstract below:

When doing the research, did you have any surprising results? 

MM: The whole project was a surprise. I guess the biggest shock was when we first detected the SARS-CoV-2 spike protein in the choroid plexus and in the cortex of haemorrhagic samples. That was the proof that there was an association between SARS-CoV-2 infection and the haemorrhages that we had observed. 

CB: It was interesting to see that the majority of the haemorrhagic samples were between 12-14 post conception weeks (pcw). The haemorrhages found in these younger samples (12-14 pcw) were more recent than the haemorrhages found in older samples (19-21 pcw). This suggests a critical window of development where viral infection could have more serious consequences on fetal brain health. Specifically, the integrity of the vasculature could be more severely affected at these younger stages (12-14 pcw) when the blood-brain barrier is still forming, making the brain more susceptible to neurovascular damage.

What were the challenges you faced when working on this project? 

MM: To me the biggest challenge was keeping all the data organised. We processed 26 different samples (plus a similar number of aged-matched controls) which came with a unique 5-digit HDBR number that we had to replace with other codes in order to make our analysis blind. Hundreds of immunofluorescence stainings, for tens of biological markers, had to be imaged generating a huge amount of data that had to be properly saved, analysed and ultimately had to be linked back to all the samples examined. 

Another challenge, given the nature of this research, was that before sharing our results we had to be completely sure that our data was correct. We had to be extra careful in analysing and evaluating our staining, using isotype control antibodies, and making sure our results were consistent with our collaborators’.

CB: Due to the limited knowledge on such a new topic, it was hard to investigate the link between SARS-CoV-2 infection and the high incidence of cortical haemorrhages. When we got started on the project, the only studies on the impact of SARS-CoV-2 infection on the brain had been carried out in cortical organoids, which lack vasculature as well as immune cells. We asked various labs with expertise in brain development and maternal infection, but nobody had ever seen such injuries in human fetal brain tissue samples. This was how we started collaborating with the Giacca, Miron and Williams labs whose contribution was key to progress in this project. 

What impact will/should your results have on public health advice? 

MM: This is an important finding, given that the Covid19 pandemic is still ongoing, and it could have an impact on public health advice. At the time it wasn’t clear if SARS-CoV-2 could be passed from mother to foetus and what the consequences of SARS-CoV-2 infection on foetal brain could be. Although, we still don’t know if these haemorrhages are the indirect result of an immune reaction from the mother or are a direct effect of the viral infection, we thought it was important to share our findings with both the scientific community and the general public, so that everyone was aware.

KL (Katie Long): As the mechanisms leading to the haemorrhages are not yet understood, we aren’t currently in a position to give public health advice, however our colleague Professor Lucilla Poston CBE, Professor of Maternal & Fetal Health at King’s College London, recommended “We know that severe viral infection may influence the fetal brain, but this important study is the first to suggest that this may occur in pregnancies affected by COVID infection. Whatever the cause, a direct effect of the virus or an indirect consequence of maternal infection, this study highlights the need for pregnant women to be vaccinated against COVID-19, thus avoiding complications for both mother and baby.”

CB: Our results should raise awareness on maternal viral infection during pregnancy and encourage pregnant women to take vaccination against SARS-CoV-2. Our study also highlights the need to better understand the risks associated with maternal SARS-CoV-2 infection and its impact on later neurological development.

Where will this story take the lab, and more broadly research in this area? 

KL: We are still receiving fetal brain samples with haemorrhages, although the incidence of this has slowed significantly after pregnant women were offered the COVID-19 vaccines. We will continue to try to understand why these haemorrhages occur and what impact they might have on the developing brain. 

What next for you after this paper?  

 MM: This was my very first paper and I am glad to have contributed to this important finding. I learnt a lot from this, and I am proud of the work we all did. As a 2^nd year PhD student my main focus now is to investigate neuronal migration disorders in the human fetal brain which is what my project was originally about. Even though I won’t have time to continue this research, as I have to prioritise my PhD project, I will be happy to help and support whoever will keep working on this. 

CB: I have now left the lab and started a PhD focusing on glioblastoma and neural stem cells. Despite the shift in research topics, working on this project has taught me a lot about academic research. Specifically, it showed me how scientific research is often nonlinear, results are sometimes unexpected, publishing can be quite complex but, ultimately, the whole process is extremely rewarding.

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