Alzheimer’s disease (AD) is a clinically and biologically complex neurodegenerative disorder, commonly manifesting with amnestic cognitive impairment, decline in language and executive function, and disturbances in motor function. Key risk factors of AD include age, female sex, certain genetic variants (such as apolipoprotein ε4), prior head injuries, educational background, metabolic syndrome, and lifestyle (e.g., diet, inactivity, and smoking) (Livingston et al., 2020). However, this broad and evolving array of factors, in addition to their temporal and individual variability, complicates efforts to determine the exact roles of these factors as causative agents, disease markers, or drivers of disease progression (Duchesne et al., 2024).

Historically, theories of AD pathogenesis have sought to provide a unifying etiological explanation. A 2024 scoping review by Duchesne et al. (2024), found that approximately three-quarters of 131 original manuscripts published since 1980 proposed unifactorial hypotheses, most commonly focusing on amyloidogenic, metabolic, infectious, and cerebrovascular/blood brain barrier (BBB) pathways. Among these, the amyloid cascade hypothesis (Hardy and Higgins, 1992) —which posits that abnormal amyloid precursor protein (APP) processing leads to excessive β-amyloid (Aβ) peptide accumulation, plaque formation, and subsequent neuronal dysfunction—has guided AD research and drug development for over three decades. However, this unifactorial theory has been increasingly revisited (Karran et al., 2011; Morris et al., 2014; Selkoe and Hardy, 2016) and, in part, reconsidered based on evidence that amyloid accumulation alone neither fully accounts for the clinical heterogeneity of AD (Jansen et al., 2015; Parent et al., 2023) nor guarantees that Aβ-targeted therapies will yield meaningful cognitive improvements (Asher and Priefer, 2022; Kim et al., 2022), although newer treatments display promise (van Dyck et al., 2023).

As our understanding of AD pathogenesis evolves, it is increasingly recognized that AD is not an isolated brain disorder, but rather the culmination of multifactorial, systemic, and chronic pathological processes affecting multiple organs and systems (Cerasuolo et al., 2023; Decourt et al., 2022; Duchesne et al., 2024). In this regard, the cardiovascular system has garnered particular interest (Scheffer et al., 2021). Evidence increasingly suggests that peripheral vascular health, blood flow, endothelial function, and blood-brain barrier integrity play integrals roles in AD pathogenesis (Nordestgaard et al., 2022; Scheffer et al., 2021; Venturelli et al., 2018). Vascular dysfunction and AD also appear to share common risk factors and potentially overlapping pathogenic mechanisms, such as Aβ production and vascular deposition, and endothelial damage contributing to microangiopathy (Nordestgaard et al., 2022; Venturelli et al., 2018). Notably, cerebral hemodynamic alterations appear early in AD and have been proposed as both diagnostic and prognostic markers (Saka et al., 2022), while similar disturbances in peripheral vasculature have been correlated with disease severity and may influence the course of AD through complex interactions with cerebral vessels (Venturelli et al., 2018). Despite these connections, the specific relationship between vascular disease and AD remains only partially elucidated, and how peripheral vascular dysfunction relates to cerebral changes and disease progression is not yet defined (Eisenmenger et al., 2023). Addressing these knowledge gaps and further investigating vascular contributions to AD’s heterogeneous etiology aligns with the expressed need for more integrative theoretical frameworks that capture the complexity of this condition (Duchesne et al., 2024), and such efforts have the potential to illuminate novel preventive, diagnostic, and therapeutic pathways (Barbera et al., 2023; Duchesne et al., 2024).

Intravital microscopy (IVM) is a sophisticated imaging method which allows for microscopy to be performed in living mice through a surgical window. This method employs light transmission, fluorescent dyes, conjugated antibodies, or genetically engineered ex vivo reporter system to system to study real-time tissue physiology (Taqueti and Jaffer, 2012). IVM can enable identification of temporal relationships between disease processes and determine cause-and-effect in responses to stimuli (Kranig et al., 2020; Stein and Gonzalez, 2017; Wissmeyer et al., 2022). Furthermore, IVM allows tissues to be examined following repeat exposures, reducing effects of biological variation between subjects, and/or experimental variation across treatments. Various IVM models have been developed to study acute and chronic vascular pathologies, such as atherosclerosis, peripheral vascular disease (PVD) and cerebral vascular dysfunction (Taqueti and Jaffer, 2012; Wissmeyer et al., 2022). This approach offers a controlled assessment of vascular function and how it relates to disease processes (Bearden et al., 2004; Kranig et al., 2020; Stein and Gonzalez, 2017; Taqueti and Jaffer, 2012; Wissmeyer et al., 2022).

Given the growing recognition that vascular dysfunction contributes to AD pathogenesis, and the identified need for multimodal preventive and therapeutic strategies (in addition to anti-amyloid treatments) (Livingston et al., 2020) exploring the relationships between peripheral vascular dysfunction and AD is necessary. In this pilot study, we investigated vasomotor reactivity in skeletal muscle of a murine model of Alzheimer’s disease (AD) using a standardized intravital microscopy (IVM) protocol. Arterioles within the gluteal muscle of APP/PS1 transgenic (Tg) and wild-type (WT) mice were exposed to vasodilatory, vasoconstrictive, and inflammatory agents to assess differences in vascular responsiveness between these two groups. We hypothesized that the arterioles of APP/PS1 transgenic mice would exhibit reduced vascular responsiveness compared to WT mice.

The procedures described below were an extension of protocols developed previously (Bearden et al., 2004).

All procedures were approved by the Institutional Animal Care and Use Committee of the University of Northern British Columbia in accordance with the Canadian Council of Animal Care (CCAC) guidelines.

This pilot study investigated vascular reactivity in Alzheimer’s disease using IVM in the APP/PS1 (line 85) mouse model (Jackson Laboratories). These mice express the Mo/APP695swe fusion protein and the PS1-dE9 variant, both of which are linked to early-onset familial Alzheimer’s disease and β-amyloid protein production. By employing intravital microscopy, we assessed differences in vasomotor responsiveness of skeletal muscle arterioles to PE, ACh, histamine, and compound 48/80 agents. No significant differences in percent diameter change were observed between WT and Tg mice in response to PE (10^–8 to 10^–5), histamine (10^–8 to 10^–4), or compound 48/80 (10^–8 to 10^–3). Histamine and compound 48/80 exhibited well-documented biphasic responses, with concentrations 10^–8 to 10^–6 of histamine and 10^–8 to 10^–6 of 48/80 resulting in vasodilation, and concentrations 10^–5 and 10^–4 of histamine and 10^–5 to 10^–3 of 48/80 resulting in vasoconstriction. Neither dilatory of constrictive responses showed significant differences between WT and Tg populations. However, it appeared vasodilatory capacity of Tg showed a significant reduction in vasodilation at an ACh concentration of 10^–5 when compared to WT, and there was a significant reduction in net diameter change among Tg mice. Furthermore, there were trends toward better constrictive and total vasoactive response among WT compared to TG mice at higher concentrations, but the differences did not reach statistical significance.

The APP/PS1 (line 85) mouse model mimics AD pathogenesis by promoting elevated β-amyloid peptide production in CNS neurons. Given the bi-directional movement of β-amyloid peptides across the BBB, an increase has been observed in the peripheral blood of APP/PS1 mice (Xiong et al., 2011). β-amyloid peptides have been shown to accumulate in vasculature, triggering inflammation and damage to vascular endothelium and smooth muscle contributing to vascular dysfunction (Anzovino et al., 2023; Koizumi et al., 2015). The findings of the current study align with previous literature showing reduced vasodilatory function in AD (Han et al., 2008; Khalil et al., 2007; Venturelli et al., 2018). Previous studies have suggested reduction in cerebral and peripheral blood flow observed in AD correlates with reduced nitrous oxide (NO) bioavailability (Morris et al., 2014). NO is a critical mediator of ACh-induced vasodilation; and is released from vascular endothelial cells following muscarinic receptor stimulation (Kellogg et al., 2005). However, endothelial-dependent NO release also contributes to the vasodilatory effects of histamine through activation of endothelial H1 receptors (Berra-Romani et al., 2019). Notably, a reduction in vascular responsiveness was not observed between Tg and WT mice with exposure to vasodilatory concentrations of histamine. While mechanisms underlying histamine- and ACh-induced vasodilation involve factors beyond NO, these findings suggest additional factors may be involved in reduced vascular responsiveness to ACh (Han et al., 2008; Khalil et al., 2007; Venturelli et al., 2018). Furthermore, lack of reduction in histamine- and compound 48/80-mediated vasodilation suggests observed reductions in vascular reactivity are potentially mediated more through direct endothelial dysfunction rather than inflammatory processes. Future research should investigate the molecular and morphological changes in vascular structures and their response to vasoactive agents. Such studies could help elucidate the mechanisms underlying AD-associated vascular dysfunction and provide insights into how AD progression affects both peripheral and cerebral vascular function.

Reductions in cerebral and peripheral blood flow have been directly associated with the progression of AD (Venturelli et al., 2018). Reduced blood flow may occur from intravascular accumulation of atherosclerotic or β-amyloid plaques, or from increased vascular resistance or resting vascular diameter. Vascular resistance is governed by the effects of vasodilatory and vasoconstrictive agents and is influenced by not only the concentration of these agents, but also the responsiveness of vasculature. It is feasible that reduced vascular responsiveness may be a potential contributor to reductions in peripheral blood flow observed in AD. This study showed a significant reduction in acetylcholine-mediated vasodilation and reduced net diameter change between Tg and WT mice. Literature has shown that elevated soluble β-amyloid levels can induce both cerebral and peripheral vasoconstriction (Arendash et al., 1999; Suo et al., 1998; Suo et al., 2000). This suggests that increased soluble peripheral β-amyloid levels may contribute to elevated resting vascular tone in AD. However, in the present study, no significant difference in resting vessel diameter was observed between Tg and WT mouse populations. One possibility is that experimental conditions, such as surgical stress and anesthetic use, may have influenced resting tone and vasoreactivity in both groups, potentially minimizing differences in responsiveness.

Despite recent advances, the relationship between peripheral and cerebrovascular dysfunction in AD remains unclear. Dysfunction of peripheral vasculature may indirectly contribute to AD progression through influences on cerebral vasculature. Peripheral vascular disease has been shown to compromise blood-brain barrier integrity through hemodynamic stress and resulting damage. This could impair cerebral β-amyloid clearance, promote cerebral accumulation, vascular deposition, and thereby compromise cerebral vasculature (Nyúl-Tóth et al., 2024; Sagare et al., 2012). Alternatively, peripheral vasculature dysfunction may contribute to sarcopenia and dyanpenia observed in AD. Studies have shown correlations between reductions in muscle mass and strength, and cerebral volume and cognitive decline, in AD (Liu et al., 2022; Ogawa et al., 2018). Peripheral vascular dysfunction could lead to hypoperfusion of skeletal muscle resulting in tissue ischemia, reduced contractility and muscle atrophy. Future research should aim to clarify the temporal relationship between cerebral and peripheral vascular dysfunction and how this relates to AD progression as well as the potential role of peripheral vasculature in motor symptoms observed in AD. These studies could help establish peripheral vasculature as a therapeutic target for the treatment of motor symptoms and possibly cognitive decline. Therefore, peripheral vascular dysfunction may hold utility as a biomarker for AD progression and its treatment could improve the quality of life of AD patients.

This study has limitations. One of the primary constraints is the limited sample size, further compounded by the exclusion of certain mice due to dropout. Furthermore, post-dose-response curve, the Tg group frequently demonstrated a prolonged return to baseline arteriolar diameter, manifesting an element of inconsistency. Enlarging the sample size in subsequent studies could offer greater precision and reduce the degree of inconsistency across experimental responses. It is also worth noting that the experiments and the recording of vessel diameters were conducted by two different observers. This could have introduced measurement bias, potentially amplifying variability in the recorded measurements. However, to minimize this source of variability, we ensured that an equivalent number of WT and Tg mice were assessed by each observer. Finally, alterations in vascular wall morphology and cellular composition, including inward or outward remodeling of arteriole vasculature were not assessed in this study, and could have independently influenced vasoreactivity. This should be assessed in future studies for a greater understanding of the mechanisms involved in reduced vascular responsiveness.

This study’s preliminary findings suggest that endothelial dysfunction underlay altered vascular responses in a murine model of AD, evidenced by significant main effects of mouse type on overall responses to acetylcholine and combined vasoactive stimuli (PE + ACh), as well as a notable difference at one ACh concentration (10^–5) and net change between resting and maximum dilation. Although many differences were not statistically significant, these initial observations lay groundwork for future studies. Moving forward, larger sample sizes, broader age ranges, and additional stressors (e.g., high-fat diets) can provide a more nuanced understanding of how AD and peripheral vasculature interact over time. Employing diverse animal models, combining intravital microscopy with histological analyses, and examining a wider range of vasoactive stimuli may help to elucidate the role of vasomotor dysfunction in AD. The inclusion of female subjects will also help clarify whether sex differences influence peripheral resistance arteries in AD and the impact of hormone differences. By extending the investigation beyond the brain, this research encourages a more integrative view of AD pathogenesis, highlighting the peripheral vasculature’s role and encouraging exploration and development of multimodal therapeutic strategies aimed at vascular targets in the fight against Alzheimer’s disease.