Angiotensin II-Mediated Neuroinflammation in the Hippocampus Contributes to Neuronal Deficits and Cognitive Impairment in Heart Failure Rats

Abstract

Background: Heart failure (HF) is a debilitating disease affecting >64 million people worldwide. In addition to impaired cardiovascular performance and associated systemic complications, most patients with HF suffer from depression and substantial cognitive decline. Although neuroinflammation and brain hypoperfusion occur in humans and rodents with HF, the underlying neuronal substrates, mechanisms, and their relative contribution to cognitive deficits in HF remains unknown.

Methods: To address this critical gap in our knowledge, we used a well-established HF rat model that mimics clinical outcomes observed in the human population, along with a multidisciplinary approach combining behavioral, electrophysiological, neuroanatomical, molecular and systemic physiological approaches.

Results: Our studies support neuroinflammation, hypoperfusion/hypoxia, and neuronal deficits in the hippocampus of HF rats, which correlated with the progression and severity of the disease. An increased expression of AT1aRs (Ang II [angiotensin II] receptor type 1a) in hippocampal microglia preceded the onset of neuroinflammation. Importantly, blockade of AT1Rs with a clinically used therapeutic drug (Losartan), and delivered in a clinically relevant manner, efficiently reversed neuroinflammatory end points (but not hypoxia ones), resulting in turn in improved cognitive performance in HF rats. Finally, we show than circulating Ang II can leak and access the hippocampal parenchyma in HF rats, constituting a possible source of Ang II initiating the neuroinflammatory signaling cascade in HF.

Conclusions: In this study, we identified a neuronal substrate (hippocampus), a mechanism (Ang II-driven neuroinflammation) and a potential neuroprotective therapeutic target (AT1aRs) for the treatment of cognitive deficits in HF.

Keywords: angiotensin II; depression; heart failure; hippocampus; microglia.

Figure 1-. Microglial activation and increased cytokine levels in the hippocampus of HF rats

A IBA1-positive microglia in the DH of ham rats. CA2/3= cornu ammonis 2/3. B Images of microglia in sham and HF rats, and their assessment of somatic volume (n=6 per group). C Quantification of microglial morphology at early and late HF stages, showing a de-ramified microglial phenotype with reduced cell volume, surface area and filament length in HF (n=8 per group). D Quantification of activated microglia based on their morphological appearance in sham and HF rats. E Sholl analysis for microglia in sham and HF rats. Heat map analysis (256 microglia, 32 per animal, 8 rats per group) reveals reduced microglial complexity in HF rats. Left numbers indicate microglial reach (in μm), color coding indicates peak Sholl values of individual microglia. F The percentage of activated microglia correlates with the severity of disease (EF%) in HF rats. p<0.05*, p<0.01** and p<0.001*** (n=8/group). Scale bars 300μm (a, top left), 25μm (a, top right), 10μm (a, bottom right), 10μm (b) and 50μm (c).

Figure 2-. Cytokine mRNA expression correlates with microglial morphology

A Quantification of cytokine mRNA levels in HF relative to sham rats (n=6 per group). B Schematic workflow used for correlative assessment of microglial complexity with cytokine mRNA levels. Images show samples of microglia negative for IL-1β (b) and positive for C1q (c). C Images of microglial IL1β, TNF-α or C1q mRNA in Sham and HF rats. Note that less complex microglia in HF rats (bottom rows) have more cytokine mRNA. Arrowheads: co-localization of IBA1 and cytokine mRNA probe. D Cytokine mRNA levels negatively correlate with microglial complexity (each dot represents a single microglia, plot is a pool of microglia obtained from n=4 rats/group). E Images of normal and hypertrophic astrocytes in sham and HF rats respectively. F Quantification of A1/A2 astrocyte mRNA markers in HF rats relative to Shams (n=6 per group). p<0.05*, p<0.01** and p<0.001***. GFAP = glial fibrillary acidic protein; GS = glutamine synthetase. Scale bars 10μm (b, c) and 20μm (e).

Figure 3-. Apoptotic signaling and altered pyramidal neuronal function in HF rats

A cCasp3-positive cells at early and late HF stages. Note the absence of cCasp3 staining in sham rats. High magnification images show nuclear deterioration and overlap of DAPI and cCasp3 signal in HF rats. B Quantification of cCasp3-positive signal at early and late HF stages (n=5/group). C Quantification of cell type-specific DAPI nuclear size. D Quantification of mean single cell neuronal cCasp3 signal in sham and HF rats (n=4/group). E Representative whole-cell voltage traces in response to depolarizing/hyperpolarizing current injection in CA1 pyramidal neurons from Sham (left) and HF (right) rats. F Mean I-V curves in sham (circles, n=11 cells/2 rats) and HF (squares, HF n=9 cells animals/ 3 rats. Note the decreased slope (input resistance) in sham rats. Input/output function plots from the same CA1 neurons in D, showing a diminished firing discharge in HF rats p<0.05*, p<0.01** and p<0.001***. Scale bars 100μm (a, overviews), 25μm (a, enlarged images) and 5μm (enlarged insets).

Figure 4-. Evidence for a hypoperfusion/hypoxic state in the hippocampus of HF rats

A Changes in hypoxia markers Hif-1α and (B) Hif-2α mRNA in the DH at early and late HF stages relative to Sham (n=5 /group). C Schematic depiction of in vivo measurement of pO₂ and cerebral blood flow (CBF) in the DH. HF rats display lower basal hippocampal pO₂ levels (n=4 sham, n=6 HF, left panel) as well as decreased basal CBF (n=5 sham, n=4 HF, right panel). D Schematic depiction of angiography via intra-carotid infusion of Rho70 dye. Rho70-labeled blood vessels within the DH of sham and HF rats. E HF rats display significantly increased vessel density. F Confocal images show vascular CD31/PECAM-1 staining in the DH of sham and HF. Note the significantly increased vascular density in HF compared to sham rats. Scale bar 100μm (e) and 200μm (f). p<0.05*, and p<0.001*** vs sham; p<0.01^## and p<0.0001^### vs HF early.

Figure 5-. Upregulation of AT1aRs in hippocampal microglia of HF rats

A Changes in AT1a mRNA levels in HF rats relative to Sham at early and late HF stages (n=5/6 rats per group). B Images of AT1aR-positive microglia (white arrowheads) in DH of sham and HF rats. Enlarged inset shows magnification of an exemplary AT1aR-positive microglia. C The number of AT1aR mRNA-positive microglia is increased in HF rats (left). AT1aR-positive microglia are less complex than AT1aR-negative microglia (right) (n=5 rats per group). D Left: Assessment of AT1aR and cytokine mRNA expression via qPCR in HF relative to Sham rats 10 days post HF surgery (n=5/group). Right: Quantification of % microglial showing a positive AT1aR mRNA expression in in sham and HF rats 10 days post-surgery via RNAScope. E Schematic depiction of intra-carotid infusion of AngII_fluo. F Images showing leakage of AngII_flu (green, following i.v. infusion) in the DH of a HF rat. Insets 1–3 (corresponding to squared areas in left panel) show co-localization of AngII_fluo with IBA1-positive microglia. Right panels: Three-dimensional reconstructions showing accumulation of AngII_fluo both in microglia processes and somata (arrowheads). Quantification of hippocampal extravasated (EV) AngII_fluo in sham and HF rats (n=5/group, left panel). Bar graphs showing an overall increase in extravasated hippocampal AngII_fluo (left), increased AngII_fluo positive microglia (middle), and increased AngII_fluo levels within individual microglial cells (right) in the DH of HF compared to sham rats (n=5/group). p<0.05*, p<0.01** and p<0.001***; p<0.01^## vs HF early . Scale bars 20μm (b), 5μm (b), 10μm (c), 100μm (e) and 10μm (f).

Figure 6-. AT1aR blockade improves neuroinflammation and cognitive performance in HF rats

A AT1aR blockade (Losartan) reduced mRNA levels of IBA1, GFAP and various cytokines, but not AT1aR, Hif-1α or Hif-2α in late stage HF rats (n=6 per group). B Images of microglia in HF rats with and without Losartan. C Assessment of microglial morphometry in HF rats with and without Losartan (n=6/group, HF late). D Losartan significantly reduced cCasp3 immunoreactivity in HF rats (n=5/group). E, HF rats subjected to Losartan displayed significantly more spontaneous alternations without changes in the total number of arm entries. F, Losartan-treated HF rats showed higher retention latency without difference in training latencies during inhibitory avoidance testing (n=19 HF, n=22 HF + Losartan). p<0.05*, p<0.01** and p<0.001*** Scale bars 25 μm (b) and 150 μm (d).