Reactive medullary astrocytes are associated with RVLM neuroinflammation in heart failure rats
Authors: Las Heras Alexandra , Díaz‐Jara Esteban , Toledo Camilo , Pereyra Katherin , Schwarz Karla G. , Diaz Hugo , Del Rio Rodrigo 
Published: May 2022

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome. Brain inflammation has been suggested to play a key role in HFpEF progression by modulating the activity of several brainstem circuits encompassing cardiovascular and respiratory regulation. Indeed, we recently reported RVLM‐C1 catecholaminergic neurons hyperactivity in rats with HFpEF. However, the precise mechanism underpinning changes in C1 activity in HFpEF remains to be determined. Interestingly, glial cell activation within the RVLM (particularly astrocytes) has been shown to modulate C1 neuron activity. Despite this evidence whether astrocytes may become activated in HFPEF has not been previously studied. Accordingly, we aimed to determine changes in astrocyte activity within the RVLM of HFpEF rats and its relation with RVLM neuroinflammatory status. Adult male Sprague‐Dawley rats (250 g) underwent volume overload to induce HFpEF. Eight weeks after surgical induction of HFpEF, rats underwent respiratory function recordings in freely moving conditions using whole‐body plethysmography. At the end of the physiological experiments, brains were harvested, fixed in 4% PFA and processed for immunohistochemical analysis. Immunofluorescence for tyrosine hydroxylase and glial fibrillary acidic protein (GFAP) were performed in 25 μm free‐floating brainstem sections containing the RVLM. Sholl analysis was performed to evaluate astrocytic morphological differences between HFpEF and Sham rats (total length of processes, # of processes, maximum intersection radius, # of intersections). RVLM neuroinflammatory status was measured in micropunches by immunoblot (GFAP) and qPCR (TNF‐α, IL‐1β). Compared to Sham rats, HFpEF animals showed clear signs of astrocyte reactivity evidenced by increases in the number of astrocyte processes (11.27 ± 1.096 vs. 18.64 ± 1.333; HFpEF vs. Sham, respectively), length of astrocyte processes (267.5 ± 17.92μm vs. 392.8 ± 22.53μm; HFpEF vs. Sham, respectively) and number of intersections (3.410 ± 0,25 vs. 4.538 ± 0.317; HFpEF vs. Sham, respectively). Consistently, we found an increased GFAP expression within the RVLM of HFpEF rats compared with Sham rats (99.9 ± 5.9 vs. 135.3 ± 15.8; HFpEF vs. Sham, respectively. In addition, we found that TNF‐α and IL‐1β were both augmented in HFpEF (~7 and ~3 fold, respectively) compared to Sham. Notably, respiratory dysfunction observed in HFpEF were significantly and positively correlated with the degree of activation of RVLM astrocytes. Indeed, respiratory irregularity score (R2 = 0.98; p<0.05), short‐term variability of the breath‐to‐breath interval (R2 = 0.98; p<0.05), and apneas/hypopneas index (R2 = 0.94; p<0.05)) were all related to GFAP expression in HFpEF rats. Our findings showed the presence of reactive astrocytes within the RVLM of HFpEF animals and that the degree of astrocyte activity was associated with RVLM neuroinflammation. Together our results suggest that increases in astrocyte reactivity in HFpEF may help to induce/maintain a pro‐iflammatory niche within the RVLM that ultimately lead to C1 neuron activity alterations.