Dysfunction of the neurovascular unit (NVU) presents a novel indicator for CNS injury after allogeneic hematopoietic stem cell transplantation

Abstract

Abstract Introduction: Graft-versus-host-disease (GVHD) is a common complication after allogeneic hematopoietic stem cell transplantation (HSCT) and is mediated by activation of both innate immune pathways and alloreactive adoptive T cell responses. It primarily affects skin, intestinal tract, and liver. 60% of the patients report cognitive dysfunction which could present toxicity or central nervous system / brain GVHD. The neurovascular unit (NVU) is a structural and functional complex that maintains homeostasis and metabolic balance in the central nervous system. The aim of this study is to noninvasively investigate the functional elements of the NVU, cerebral blood flow (CBF), vascular and metabolic changes, using 7T-magnetic resonance imaging (MRI) using a murine acute GVHD (aGVHD) model. Methods: Recipient B6D2F1 received conditioning with total body irradiation at a total dose of 12Gy followed by infusion of a cell mixture of 5 million bone marrow cells and 6 million splenocytes from either syngeneic B6D2F1 or allogeneic C57BL/6 donors via tail vein injection. Animals were imaged with a 7T MRI scanner under 1.5% isoflurane anesthesia on days 7, 14, 28, and 42 post-transplantation and compared to age-matched untreated controls. Images were analyzed using Segment v4.0 (Medviso), ITK-SNAP software to quantify volumetric changes and perfusion in the different brain regions. Mice were monitored for clinical signs of GVHD, survival and analyzed on day +42. Immunofluorescence and western blot analysis were performed using collected brain tissue lysates to study markers of neurovascular unit components including glial markers, neurons and endothelial cells. Results: The allogenic group showed reduced survival and significant difference in clinical GVHD score (p<0.0001) starting on day +21 when compared to the syngeneic groups. Already before that, 7T-MRI imaging analysis showed significant cerebral blood flow (CBF) hypoperfusion in allogeneic group on day 7 (cortex: p=0.0045, hippocampus: p=0.03378) and day 14 (cortex: p<0.0001, hippocampus: p=0.0047). Structural analysis showed a significant percentage of reduction in hippocampal volume from day 7 to day 42 in the allogenic group compared to the syngeneic group (p=0.04). Allogeneic recipients showed increased microglial activation (Iba1+) and significantly increased expression of GFAP (p=0.02), correlating with reactive astrocytes and infiltration of CD3+ T-cells along with reduced expression of neuronal marker (NeuN) in cortex and hippocampus regions. Conclusion: Our results suggest regional structural abnormalities and reduced CBF perfusion involving structures of cognition and memory along with alterations in neurovascular unit components after allogeneic HSCT, potentially providing the stage for neurotoxicity and cognitive defects, and allowing for the development of novel preventive and therapeutic interventions.