Background
Hypertension is a serious public health issue, being highly prevalent in the elderly [
1] and a leading risk factor for stroke [
2], cognitive impairment, and dementia [
3]. Despite the vast array of drugs to lower blood pressure, hypertension control remains suboptimal stressing the need for better therapeutic management.
Besides reflecting an increase in arterial blood pressure, hypertension consists of a chronic, low-grade inflammation involving innate and adaptive immune cells [
4]. The link between systemic inflammation and high blood pressure has been well described by human [
5‐
7] and animal data [
8‐
14]. However, the effects of hypertension on the pathogenesis of cerebral inflammation are less understood.
Studies examining the role of cerebral inflammation in experimental hypertension have focused on the paraventricular nucleus (PVN), a region of the hypothalamus involved in the sympathetic control of blood pressure [
15]. Using anti-inflammatory approaches (with minocycline or IL-10 administered directly to the brain) or depleting microglia by intra-cerebro-ventricular administration of diphtheria toxin (DT) to transgenic CD11b-DT receptor mice, these studies have shown that inflammation and microglia play a central role in the development of high blood pressure in mice and rats [
16,
17]. While finding that inflammation influenced blood pressure, they also showed that hypertension, which was achieved via systemic infusion of angiotensin II (Ang II), was capable of producing increases in microglial density, soma enlargement, and reductions in microglial process length, as well as greater production of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in this brain region [
16‐
18].
Given the strong associations between mid-life hypertension and late-life dementia [
19‐
21], the involvement of other vulnerable brain structures such as the hippocampus, a key region involved in memory formation, is also of significant interest. Likewise, whether the cerebral inflammatory effects of circulating Ang II are a consequence of its own cellular actions only, whether they are mediated by its increase in blood pressure, or whether blood pressure itself can do so independently of Ang II remains unclear.
Therefore, this study was set to answer these questions. We administered Ang II in the systemic circulation of C57BL/6 mice during 7 or 14 days and examined the inflammatory response in the whole brain and in the hippocampus using a multidisciplinary approach. In order to dissect the effects of Ang II and blood pressure, we delivered pressive versus subpressive Ang II doses and phenylephrine, or used the peripheral vasodilator hydralazine to prevent hypertension. Our results show that Ang II and blood pressure can differentially contribute to the development of hippocampal inflammation in mice.
Discussion
This study presents several novel findings. First, it provides new knowledge on specific brain regions involved in Ang II-induced inflammation beyond the PVN, by showing increased TNF-α production, elevated GFAP expression, and microgliosis in the hippocampus. Second, it presents a thorough quantitative characterization of hippocampal microglia morphology achieved via EM, providing a better understanding of their functional activation state in relation to hypertension. Third, it reveals that both increased blood pressure and Ang II concentrations are important contributors to hippocampal inflammation induced by systemic Ang II.
Ang II, the main effector of the renin-angiotensin system, is a circulating peptide hormone and a potent vasoconstrictor that exerts key roles in the regulation of blood pressure and the development of hypertension [
24]. The brain also has its own intrinsic renin-angiotensin system from where Ang II is produced [
25‐
28]. In rodents and humans, Ang II binding sites are expressed widely throughout the brain including the cerebral vasculature and circumventricular organs (CVO), where they have access to systemic Ang II [
27,
29,
30]. Within the brain parenchyma, angiotensin II type I (AT1) receptors are highly expressed in neurons among specific brain regions involved in autonomic and cardiovascular regulation and, although in lower levels, in other areas including the cerebral cortex, hippocampus, basal ganglia, and cerebellum [
27,
29,
30].
Whether systemic Ang II and increased blood pressure contributed to cerebral inflammation beyond the cerebral vasculature and PVN was not clear before this study. Therefore, we delivered pressive and subpressive doses of Ang II and used phenylephrine or hydralazine, which modulate blood pressure independently of the renin angiotensin system (phenylephrine increases blood pressure via α-1 adrenergic receptors, and hydralazine lowers blood pressure by relaxing vascular smooth muscle). With these approaches, our results show that circulating Ang II leads to hippocampal inflammation and cerebral gliosis in a dose-dependent manner and that increased blood pressure is an important contributor to the brain pro-inflammatory effects of Ang II in mice.
Is blood pressure elevation alone sufficient? With administration of phenylephrine, we did not observe an increase in Iba-1, as we have seen with the highest hypertensive dose of Ang II, even though the dose of phenylephrine was ten times higher than that of Ang II (19 μg/kg/min versus 1900 ng/kg/min). In addition, analysis showed a non-linear correlation between hippocampal TNF-α and blood pressure, suggesting that other factors could modulate the association between these parameters. For example, the breakdown of the blood-brain barrier (BBB) induced by Ang II may be involved in the inflammatory process [
31,
32]. Indeed, a study from Marvar showed that Ang II is critical for BBB damage, independently of blood pressure [
31]. The effects of Ang II are also highlighted in this study by the fact that hippocampal Iba-1 levels rose with increasing concentrations of Ang II, while the increase in blood pressure produced by these hypertensive doses was similar. Likewise, increasing subpressive Ang II (200 ng/kg/min) infusion duration to 14 days resulted in a significant increase in hippocampal CD68 mRNA, with a similar trend for Iba-1, without changes of GFAP or TNF-α. These results emphasize the importance of Ang II actions and also suggest that a certain threshold of blood pressure may be required for the full expression of cerebral inflammation.
Despite bypassing the renin-angiotensin system, we observed a significant increase in GFAP expression in the hippocampus with phenylephrine treatment. This vulnerability of astrocytes is not surprising considering their anatomical proximity to cerebral blood vessels as part of the neurovascular unit [
33], being close targets of the pressor stress suffered by the vasculature in hypertension. Indeed, it is known that astrocytes can act as “sensors” to vascular damage, by expressing ion channels sensitive to shear stress, such as TRPV4 (transient receptor potential vanilloid subtype 4) [
34], which when activated lead to the development of astrogliosis [
35].
Beyond our study, a critical role for Ang II on cerebral inflammation is supported by a previous work showing that inhibition of AT1 receptors with candesartan, a centrally acting angiotensin receptor blocker, leads to reductions in brain inflammation induced by LPS or stroke in normotensive rats [
36,
37]. At the same time, our results on blood pressure complement the findings of Marvar and colleagues who used a similar approach with hydralazine to demonstrate that the pressor effects of Ang II are critical for the activation of circulating T lymphocytes and the expression of vascular inflammation in mice [
31]. Interestingly, there is also evidence that hydralazine attenuates cardiac fibrosis and inflammation induced by pressive Ang II in mice, suggesting that blood pressure is also a key contributor to the expression of inflammation in other end organs affected by hypertension besides the brain [
38].
With respect to hydralazine, there have been reports indicating it exhibits antioxidant properties beyond its well-recognized antihypertensive effect, including scavenger actions on peroxynitrite and oxygen radicals [
39‐
41]. Although it can be argued that attenuation of oxidative stress by this peripheral vasodilator could have helped diminish the cerebral inflammation caused by hypertensive Ang II, some considerations reaffirm its main mechanism of action through blood pressure reduction. First, the range of hydralazine doses with antioxidant properties previously reported was much higher than the dose used in this study (≥ 250 versus 150 mg/L, respectively). Second, although low hydralazine doses (similar or lower than this study) demonstrated ROS scavenging actions in vitro (reductions of oxidative radicals by 25%), this effect was significantly lower compared to classical antioxidants such as ascorbic acid, which reduced oxygen radical production by 80% [
39]. Third, in the discussed studies, the antioxidant actions of hydralazine have been evaluated in vitro in peritoneal macrophages, cultured smooth muscle cells, and isolated vessels, or ex vivo in isolated rabbit aortas. Therefore, to the best of our knowledge, the cerebral antioxidant actions of hydralazine remain to be demonstrated.
Previous studies examining the cerebral pro-inflammatory effects of pressive Ang II focused on the PVN, a region of the hypothalamus involved in the sympathetic control of blood pressure [
15]. These studies showed changes in microglial morphology (enlarged soma, process retraction) and increased production of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) [
16‐
18]. Our results confirm that pressive Ang II leads to cerebral gliosis and extends this finding to another brain region, namely the hippocampus. Our work also complements the study of Toth and colleagues who demonstrated that the hippocampal expression of the chemokines MCP-1 (CCL2) and IP-10 (CXCL10) is increased in young mice (3 months) receiving pressive doses of Ang II (1000 ng/kg/min, 28 days), a response that is exacerbated by aging (24 months), where upregulation of CD68 and TNF-α then became evident [
42].
Besides an upregulation of TNF-α and astrogliosis, we report significant morphological changes of microglia in the hippocampus of mice infused with pressive Ang II, as detected by immuno-EM. Changes in microglial morphology are informative of their functional activation state in response to stimuli or injury. In this study, we labeled microglia with Iba-1, which is a well-recognized marker specifically expressed by resident microglia and macrophages [
43,
44]. We report significant changes in quantitative shape and functional descriptors including increases in microglial process area and perimeter, reductions in circularity and solidity, and a greater number of vacuoles and increased extracellular digestion.
The increase in process area and perimeter would indicate that they are larger in the presence of Ang II, reflecting process thickening and therefore increased metabolic or lysosomal activity. This is also supported by the finding of reduced solidity and thus of a more ruffled morphology, which could be a sign of increased process motility reflecting their capacity to reorient towards sites of injury [
45]. Furthermore, the changes in vacuoles and increased extracellular digestion suggest an involvement of microglia in synaptic remodeling in the presence of Ang II. This is supported by the EM micrographs showing microglial processes with complex morphologies making contacts with pre-synaptic axon terminals, synaptic clefts, and post-synaptic dendritic spines and containing cellular debris being degraded. A seminal study recently showed that the physiological functions of microglia that prune excess synapses during development are aberrantly activated in Alzheimer’s disease animal models, contributing to synaptic loss, which is a strong correlate of cognitive decline [
46]. It is therefore possible that microglia in the presence of Ang II undergo phenotypic changes that lead to inflammation, synaptic pruning, and ultimately cognitive impairments. Indeed, these specific changes in the hippocampus are in line with our earlier findings that pressive Ang II leads to cognitive dysfunctions in tasks that are dependent on this region [
22]. They are also consistent with a recent study examining the impact of Ang II (1000 ng/kg/min, 28 days perfusion) on synaptic plasticity in mice, showing that systemic Ang II leads to reductions in hippocampal long-term potentiation, decreased density of hippocampal synapses, and reduced expression of key genes involved in synaptic plasticity, such as
Bdnf and
Homer1 [
47]. We could thus speculate that the development of hippocampal inflammation and synaptic dysfunction is a mechanistic link between early hypertension and the brain deficits that lead to neuronal injury and dementia in late-life [
19‐
21].
It is interesting that such significant and specific changes in hippocampal microglia morphology were detected in the absence of increased expression of Iba-1 or CD68 in this region. These results highlight the sensitivity of immuno-EM ultrastructural studies to reveal alterations in microglia morphological dynamics that may otherwise appear “normal” when examined by protein or gene expression analysis. With respect to other regions, the increase in Iba-1 in the whole brain in response to pressive Ang II could be a signal of increased microglial numbers or phenotypic alterations taking place outside the hippocampus (e.g., the PVN, as previously shown) or even in the white matter, an interesting hypothesis warranting future investigations.
In addition to maintaining brain homeostasis, microglia undergo phenotypic transformation upon tissue injury, or in response to Ang II, leading to the secretion of inflammatory and oxidative molecules that contribute to neurodegeneration [
48]. A recent study demonstrated that isolated hypothalamic microglia actually respond to Ang II through AT1 and TLR4 receptors, the latter which is required for the expression of oxidative damage induced by Ang II in mice [
49]. Although the role of other cells including neurons and astrocytes cannot be discarded, the ultrastructural morphological changes along with the increased hippocampal TNF-α production suggest the emergence of a pro-inflammatory microglial phenotype in mice with chronic Ang II administration. This is supported by Shen and colleagues who showed that the increase in hypothalamic TNF-α that results from in vivo Ang II administration returned to normal levels when microglia were depleted from the brain [
17].
Besides affecting microglia, our finding of increased hippocampal GFAP expression in response to pressive Ang II indicates that astrocyte activation is also involved in Ang II-induced neuroinflammation. Indeed, in a very comprehensive study, Stern and colleagues recently showed that astrocytes are critical and direct cellular mediators of Ang II actions on PVN neurons, leading to increased sympathetic outflow and to increased blood pressure [
50].
Therefore, it is likely that the cerebral inflammation induced by Ang II involves multiple pathways and cellular players, including but not limited to (i) the direct effects of circulating Ang II on AT1 receptors in cerebral vessels [
51]; (ii) the damage to the BBB [
32], which could facilitate the entry of Ang II to brain regions beyond the CVO network (i.e., the hippocampus); (iii) the consequent effects on microglia and astrocytes, leading to the expression of pro-inflammatory cytokines within the brain; and (iv) the entry of activated immune cells from the periphery.
The findings of our study must be considered within its limitations. One lesson learned from our work is that microglia morphology analysis via EM is able to detect subtler and even more informative changes than immunofluorescence, as seen by the lack of Iba-1 increase with the dose of 1000 ng/kg/min Ang II in parallel with significant changes in the morphology of microglia. In this regard, although Iba-1 levels in phenylephrine-treated animals were comparable to controls, we cannot discard the possibility that phenylephrine could have affected microglia morphology in this group. It would be interesting to examine whether metabolic changes in microglia differ upon Ang II or phenylephrine treatment despite similar effects on blood pressure. Also, our study examined only male mice and potential sex differences in the response to the pressive and inflammatory effects of Ang II cannot be discarded. Despite these considerations, this work broadens our understanding of the in vivo effects of Ang II actions in relation to hypertension and its impact on brain inflammation.