The effects of time restricted feeding on age-related changes in the mouse retina.

Dietary modifications such as caloric restriction (CR) and intermittent fasting (IF) have gained popularity due to their proven health benefits in aged populations. In time restricted feeding (TRF), a form of intermittent fasting, the amount of time for food intake is regulated without restricting the caloric intake. TRF is beneficial for the central nervous system to support brain health in the context of aging. Therefore, we here ask whether TRF also exerts beneficial effects in the aged retina. We compared aged mice (24 months) on a TRF paradigm (access to food for six hours per day) for either 6 or 12 months against young control mice (8 months) and aged control mice on an ad libitum diet. We examined changes in the retina at the functional (electroretinography), structural (histology and fluorescein angiograms) and molecular (gene expression) level. TRF treatment showed amelioration of age-related reductions in both scotopic and photopic b-wave amplitudes suggesting benefits for retinal interneuron signaling. TRF did not affect age-related signs of retinal inflammation or microglial activation at either the molecular or histological level. Our data indicate that TRF helps preserve some aspects of retinal function that are decreased with aging, adding to our understanding of the health benefits that altered feeding patterns may confer.

Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Novel intravital approaches to quantify deep vascular structure and perfusion in the aging mouse brain using ultrasound localization microscopy (ULM).

Intra-vital visualization of deep cerebrovascular structures and blood flow in the aging brain has been a difficult challenge in the field of neurovascular research, especially when considering the key role played by the cerebrovasculature in the pathogenesis of both vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). Traditional imaging methods face difficulties with the thicker skull of older brains, making high-resolution imaging and cerebral blood flow (CBF) assessment challenging. However, functional ultrasound (fUS) imaging, an emerging non-invasive technique, provides real-time CBF insights with notable spatial-temporal resolution. This study introduces an enhanced longitudinal fUS method for aging brains. Using elderly (24-month C57BL/6) mice, we detail replacing the skull with a polymethylpentene window for consistent fUS imaging over extended periods. Ultrasound localization mapping (ULM), involving the injection of a microbubble (<<10 µm) suspension allows for recording of high-resolution microvascular vessels and flows. ULM relies on the localization and tracking of single circulating microbubbles in the blood flow. A FIJI-based analysis interprets these high-quality ULM visuals. Testing on older mouse brains, our method successfully unveils intricate vascular specifics even in-depth, showcasing its utility for longitudinal studies that require ongoing evaluations of CBF and vascular aspects in aging-focused research.

Longitudinal detection of gait alterations associated with hypertension-induced cerebral microhemorrhages in mice: predictive role of stride length and stride time asymmetry and increased gait entropy.

Cerebral microhemorrhages (CMHs) are of paramount importance as they not only signify underlying vascular pathology but also have profound implications for cognitive function and neurological health, serving as a critical indicator for the early detection and management of vascular cognitive impairment (VCI). This study aimed to investigate the effects of hypertension-induced CMHs on gait dynamics in a mouse model, focusing on the utility of advanced gait metrics as sensitive indicators of subclinical neurological alterations associated with CMHs. To induce CMHs, we employed a hypertensive mouse model, using a combination of Angiotensin II and L-NAME to elevate blood pressure, further supplemented with phenylephrine to mimic transient blood pressure fluctuations. Gait dynamics were analyzed using the CatWalk system, with emphasis on symmetry indices for Stride Length (SL), Stride Time (ST), and paw print area, as well as measures of gait entropy and regularity. The study spanned a 30-day experimental period, capturing day-to-day variations in gait parameters to assess the impact of CMHs. Temporary surges in gait asymmetry, detected as deviations from median gait metrics, suggested the occurrence of subclinical neurological signs associated with approximately 50% of all histologically verified CMHs. Our findings also demonstrated that increases in gait entropy correlated with periods of increased gait asymmetry, providing insights into the complexity of gait dynamics in response to CMHs. Significant correlations were found between SL and ST symmetry indices and between these indices and the paw print area symmetry index post-hypertension induction, indicating the interdependence of spatial and temporal aspects of gait affected by CMHs. Collectively, advanced gait metrics revealed sensitive, dynamic alterations in gait regulation associated with CMHs, resembling the temporal characteristics of transient ischemic attacks (TIAs). This underscores their potential as non-invasive indicators of subclinical neurological impacts. This study supports the use of detailed gait analysis as a valuable tool for detecting subtle neurological changes, with implications for the early diagnosis and monitoring of cerebral small vessel disease (CSVD) in clinical settings.

Young blood-mediated cerebromicrovascular rejuvenation through heterochronic parabiosis: enhancing blood-brain barrier integrity and capillarization in the aged mouse brain.

Age-related cerebromicrovascular changes, including blood-brain barrier (BBB) disruption and microvascular rarefaction, play a significant role in the development of vascular cognitive impairment (VCI) and neurodegenerative diseases. Utilizing the unique model of heterochronic parabiosis, which involves surgically joining young and old animals, we investigated the influence of systemic factors on these vascular changes. Our study employed heterochronic parabiosis to explore the effects of young and aged systemic environments on cerebromicrovascular aging in mice. We evaluated microvascular density and BBB integrity in parabiotic pairs equipped with chronic cranial windows, using intravital two-photon imaging techniques. Our results indicate that short-term exposure to young systemic factors leads to both functional and structural rejuvenation of cerebral microcirculation. Notably, we observed a marked decrease in capillary density and an increase in BBB permeability to fluorescent tracers in the cortices of aged mice undergoing isochronic parabiosis (20-month-old C57BL/6 mice [A-(A)]; 6 weeks of parabiosis), compared to young isochronic parabionts (6-month-old, [Y-(Y)]). However, aged heterochronic parabionts (A-(Y)) exposed to young blood exhibited a significant increase in cortical capillary density and restoration of BBB integrity. In contrast, young mice exposed to old blood from aged parabionts (Y-(A)) rapidly developed cerebromicrovascular aging traits, evidenced by reduced capillary density and increased BBB permeability. These findings underscore the profound impact of systemic factors in regulating cerebromicrovascular aging. The rejuvenation observed in the endothelium, following exposure to young blood, suggests the existence of anti-geronic elements that counteract microvascular aging. Conversely, pro-geronic factors in aged blood appear to accelerate cerebromicrovascular aging. Further research is needed to assess whether the rejuvenating effects of young blood factors could extend to other age-related cerebromicrovascular pathologies, such as microvascular amyloid deposition and increased microvascular fragility.

IGF1R deficiency in vascular smooth muscle cells impairs myogenic autoregulation and cognition in mice.

Introduction: Cerebrovascular pathologies contribute to cognitive decline during aging, leading to vascular cognitive impairment and dementia (VCID). Levels of circulating insulin-like growth factor 1 (IGF-1), a vasoprotective hormone, decrease during aging. Decreased circulating IGF-1 in animal models leads to the development of VCID-like symptoms, but the cellular mechanisms underlying IGF-1-deficiency associated pathologies in the aged cerebrovasculature remain poorly understood. Here, we test the hypothesis that vascular smooth muscle cells (VSMCs) play an integral part in mediating the vasoprotective effects of IGF-1. Methods: We used a hypertension-based model of cerebrovascular dysfunction in mice with VSMC-specific IGF-1 receptor (Igf1r) deficiency and evaluated the development of cerebrovascular pathologies and cognitive dysfunction. Results: VSMC-specific Igf1r deficiency led to impaired cerebral myogenic autoregulation, independent of blood pressure changes, which was also associated with impaired spatial learning and memory function as measured by radial arm water maze and impaired motor learning measured by rotarod. In contrast, VSMC-specific IGF-1 receptor knockdown did not lead to cerebral microvascular rarefaction. Discussion: These studies suggest that VSMCs are key targets for IGF-1 in the context of cerebrovascular health, playing a role in vessel stability alongside other cells in the neurovascular unit, and that VSMC dysfunction in aging likely contributes to VCID.

Vascular smooth muscle cell-specific Igf1r deficiency exacerbates the development of hypertension-induced cerebral microhemorrhages and gait defects.

Cerebrovascular fragility and cerebral microhemorrhages (CMH) contribute to age-related cognitive impairment, mobility defects, and vascular cognitive impairment and dementia, impairing healthspan and reducing quality of life in the elderly. Insulin-like growth factor 1 (IGF-1) is a key vasoprotective growth factor that is reduced during aging. Circulating IGF-1 deficiency leads to the development of CMH and other signs of cerebrovascular dysfunction. Here our goal was to understand the contribution of IGF-1 signaling on vascular smooth muscle cells (VSMCs) to the development of CMH and associated gait defects. We used an inducible VSMC-specific promoter and an IGF-1 receptor (Igf1r) floxed mouse line (Myh11-CreERT2 Igf1rf/f) to knockdown Igf1r. Angiotensin II in combination with L-NAME-induced hypertension was used to elicit CMH. We observed that VSMC-specific Igf1r knockdown mice had accelerated development of CMH, and subsequent associated gait irregularities. These phenotypes were accompanied by upregulation of a cluster of pro-inflammatory genes associated with VSMC maladaptation. Collectively our findings support an essential role for VSMCs as a target for the vasoprotective effects of IGF-1, and suggest that VSMC dysfunction in aging may contribute to the development of CMH.

Microvascular smooth muscle cells exhibit divergent phenotypic switching responses to platelet-derived growth factor and insulin-like growth factor 1.

OBJECTIVE: Vascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. METHODS: We used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. RESULTS: PDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-ß and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. CONCLUSIONS: These studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.

Rejuvenation of cerebromicrovascular function in aged mice through heterochronic parabiosis: insights into neurovascular coupling and the impact of young blood factors.

Age-related impairment of neurovascular coupling (NVC; "functional hyperemia") is a critical factor in the development of vascular cognitive impairment (VCI). Recent geroscience research indicates that cell-autonomous mechanisms alone cannot explain all aspects of neurovascular aging. Circulating factors derived from other organs, including pro-geronic factors (increased with age and detrimental to vascular homeostasis) and anti-geronic factors (preventing cellular aging phenotypes and declining with age), are thought to orchestrate cellular aging processes. This study aimed to investigate the influence of age-related changes in circulating factors on neurovascular aging. Heterochronic parabiosis was utilized to assess how exposure to young or old systemic environments could modulate neurovascular aging. Results demonstrated a significant decline in NVC responses in aged mice subjected to isochronic parabiosis (20-month-old C57BL/6 mice [A-(A)]; 6 weeks of parabiosis) when compared to young isochronic parabionts (6-month-old, [Y-(Y)]). However, exposure to young blood from parabionts significantly improved NVC in aged heterochronic parabionts [A-(Y)]. Conversely, young mice exposed to old blood from aged parabionts exhibited impaired NVC responses [Y-(A)]. In conclusion, even a brief exposure to a youthful humoral environment can mitigate neurovascular aging phenotypes, rejuvenating NVC responses. Conversely, short-term exposure to an aged humoral milieu in young mice accelerates the acquisition of neurovascular aging traits. These findings highlight the plasticity of neurovascular aging and suggest the presence of circulating anti-geronic factors capable of rejuvenating the aging cerebral microcirculation. Further research is needed to explore whether young blood factors can extend their rejuvenating effects to address other age-related cerebromicrovascular pathologies, such as blood-brain barrier integrity.