Introduction

Age-related impairment of neurovascular coupling (NVC; or “functional hyperemia”) contributes to the pathogenesis of vascular cognitive impairment (VCI) [1]. Neurovascular dysfunction compromises adjustment of cerebral blood flow to the increased needs of active brain regions, impairing energy and oxygen delivery to the firing neurons and hindering washout of toxic metabolic by-products [1]. Neurovascular coupling depends on a tightly controlled interaction of activated neurons and astrocytes and the release of vasodilator metabolites from the astrocyte end-feet and microvascular endothelial cell, which elicit vasodilation in precapillary arterioles. The cellular mechanisms by which aging impairs neurovascular coupling responses primarily involve a significant reduction in endothelial production/release of nitric oxide (NO) [2,3,4].

Insulin-like growth factor-1 (IGF-1) is an anabolic hormone produced by the liver, which exerts multifaceted vasoprotective and anti-geronic effects [1, 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31]. Circulating IGF-1 significantly decreases with age in humans and in laboratory animals due to an age-related decline in GH production/release [12, 30, 32,33,34,35]. Importantly, previous studies demonstrate that circulating IGF-1 deficiency in transgenic mouse models impairs neurovascular coupling responses, mimicking the aging phenotype [9, 36]. Each cell type of the neurovascular unit (including neurons, astrocytes, endothelial cells) abundantly express IGF1R, the receptor for IGF-1 and the specific roles of IGF1R signaling in endothelial cells in regulation of NVC responses remains to be determined.

The present study was designed to experimentally test the hypotheses that IGF1R signaling modulates endothelium-dependent NVC responses in the brain and that disruption of IGF1R signaling specifically in endothelial cells impairs functional hyperemia, mimicking aspects of the aging phenotype. To test our hypotheses, we used a novel mouse model with adult-onset, endothelial cells-specific disruption of IGF1R signaling using Cre-lox technology (VE-Cadherin-CreERT2/Igf1rf/f). To assess endothelial NO-mediated NVC responses, increases in cerebral blood flow in the somatosensory whisker barrel cortex in response to contralateral whisker stimulation were measured using laser speckle contrast imaging before and after administration of an NO synthase inhibitor.

Methods

Animals

Igf1rf/f (B6;129-Igf1rtm2Arge/J; loxP sites flanking exon 3) and VE-Cadherin-Cre ERT2 (B6.FVB-Tg(Cdh5-cre)7Mlia/J; Stock No: 006137) mice were obtained from Jackson laboratories. Mice were housed (3–4 per cage) in Allentown XJ cages with Anderson's Enrich-o-cob bedding (Maumee, OH). Igf1rf/f mice were bred in house to generate experimental cohorts. Animals were housed under specific pathogen-free (including helicobacter and parvovirus free) barrier conditions in the Rodent Barrier Facility at University of Oklahoma Health Sciences Center. Mice were bred on a 14-h light/10-h dark cycle and weaned mice were maintained in a 12-h light/12-h dark cycle at 21 °C and were given access to standard irradiated bacteria-free rodent chow (5053 Pico Lab, Purina Mills, Richmond, IN) and reverse osmosis filtered water ad libitum. Male VE-Cadherin-CreERT2 mice were bred with female Igf1rf/f mice to generate VE-Cadherin-CreERT2/Igf1r+/− males, which were bred with Igf1rf/f female mice to obtain the founder colony of VE-Cadherin-CreERT2/Igf1r homozygous floxed mice, following our previously described protocol [18]. These mice were bred with Igf1rf/f mice to generate experimental cohorts of VE-Cadherin-CreERT2/Igf1rf/f and Cre-/Igf1rf/f control mice. Three-month-old mice were injected intraperitoneally with tamoxifen (75 mg/kg body weight; dissolved in corn oil) or vehicle (corn oil) for 5 days. Experiments were conducted after a period of 2 months. All procedures were approved by the Institutional Animal Use and Care Committee of the University of Oklahoma Health Sciences Center.

Measurement of neurovascular coupling responses

On the day of experimentation, mice in each group were anesthetized with isoflurane (4% induction and 1% maintenance), endotracheally intubated and ventilated (MousVent G500; Kent Scientific Co, Torrington, CT). A thermostatic heating pad (Kent Scientific Co, Torrington, CT) was used to maintain rectal temperature at 37 °C [37]. End-tidal CO2 was controlled between 3.2% and 3.7% to keep blood gas values within the physiological range, as described [9, 38, 39]. The right femoral artery was canulated for arterial blood pressure measurement (Living Systems Instrumentations, Burlington, VT) [37]. The blood pressure was within the physiological range throughout the experiments (90–110 mmHg). Mice were immobilized and placed on a stereotaxic frame (Leica Microsystems, Buffalo Grove, IL), the scalp and periosteum were pulled aside and the skull was gently thinned using a dental drill while cooled with dripping buffer. A laser speckle contrast imager (Perimed, Järfälla, Sweden) was placed 10 cm above the thinned skull, and to achieve the highest CBF response the right whiskers were stimulated for 30 s at 5 Hz from side to side as described [40, 41]. Differential perfusion maps of the brain surface were captured. Changes in CBF were assessed above the left barrel cortex in six trials in each group, separated by 5–10 min intervals. To assess the role of NO mediation, CBF responses to whisker stimulation were repeated 15 min after intravenous administration of the nitric oxide synthase inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME). Changes in CBF were averaged and expressed as percent (%) increase from the baseline value [42]. All drugs used in this study were purchased from Sigma-Aldrich (St Louis, MO) unless otherwise indicated.

Statistical analysis

Statistical analysis was carried out by unpaired t test or one-way ANOVA followed by Bonferroni multiple comparison test, as appropriate, using Prism 5.0 for Windows (Graphpad Software, La Jolla, CA). A p value less than 0.05 was considered statistically significant. Data are expressed as mean ± S.E.M.

Results

Endothelium-specific disruption of IGF-1/IGF1R signaling impairs neurovascular coupling

Increases in CBF in the somatosensory whisker barrel cortex in response to contralateral whisker stimulation were significantly attenuated in 6-month-old VE-Cadherin-CreERT2/Igf1rf/f mice (Fig. 1A–C), indicating that endothelium-specific disruption of IGF1R signaling leads to neurovascular dysfunction (n = 6–10 ♂ mice in each group).

Fig. 1
figure 1

Endothelium-specific disruption of IGF-1/IGF1R signaling impairs neurovascular coupling responses in mice. A) Representative pseudocolour laser speckle flowmetry maps of baseline CBF (upper row; shown for orientation purposes) and CBF changes in the whisker barrel field relative to baseline during contralateral whisker stimulation (bottom row, right oval, 30 s, 5 Hz) in control and VE-Cadherin-CreERT2/Igf1rf/f mice before and after administration of the NO synthase inhibitor L-NAME. B shows the time-course of CBF changes after the start of contralateral whisker stimulation (horizontal bars). Summary data are shown in C. Data are mean ± S.E.M. (n = 6–10 ♂ mice in each group), *P < 0.05 vs. Control; #P < 0.05 vs. untreated. n.s.: not significant

Full size image

Upon activation by neuronal-derived glutamate astrocytes release ATP, which elicits endothelial NO-mediated microvascular dilation in the brain [43]. Endothelial NO mediation is also critical for the upstream conduction and spreading of microvascular dilation [3]. Consistent with this concept we found that in control animals administration of the NO synthase inhibitor L-NAME (Fig. 1B–C) significantly decreased functional hyperemia in the barrel cortex elicited by contralateral whisker stimulation.

In VE-Cadherin-CreERT2/Igf1rf/f mice, the effects of L-NAME (Fig. 1B–C) were significantly decreased, suggesting that endothelium-specific disruption of IGF1R signaling impairs the endothelial NO-dependent component of NVC responses.

Discussion

Endothelial NO mediation plays a critical role both in NVC responses and the upstream conduction and spreading of microvascular dilation [3, 43]. IGF-1 receptors are abundantly expressed on endothelial cells [44]. The present study provides critical evidence that cell-specific disruption of IGF1R signaling in endothelial cells alters their function, impairing NO-mediated NVC responses. These new findings extend the results of our previous studies showing that circulating IGF-1 deficiency also impairs the endothelium-dependent NVC responses [9]. The likely mechanisms by which disruption of endothelial IGF-1/IGF1R signaling impairs NO-mediated NVC responses may include decreased NO bioavailability due to increased production of reactive oxygen species (ROS) [9]. There is strong evidence linking impaired NVC responses to impaired performance on cognitive tasks [1, 38,39,40,41, 45]. Thus, further studies are warranted to determine how the neurovascular phenotype caused by disruption of endothelial IGF-1/IGF1R signaling impacts cognitive function in mice.

Previous studies showed that in addition to regulating vasodilator function IGF-1/IGF1R signaling also modulates many other important aspects of endothelial function, including angiogenesis and barrier function [22, 23, 30, 46,47,48,49,50,51]. There is evidence that disruption of IGF-1/IGF1R signaling may also impact these aspects of cerebromicrovascular endothelial cell function, which may contribute to microvascular rarefaction and blood–brain barrier disruption, exacerbating cognitive impairment associated with IGF-1 deficiency [8, 10, 52, 53]. Circulating insulin at physiological concentrations has low affinity IGF-1R, while under experimental conditions, at supraphysiological levels, it was found that insulin and IGF-1 cross-react with each other’s receptors, albeit at a significantly lower affinity than with their own receptors. Previous studies suggested that IGF1R can regulate insulin sensitivity and NO bioavailability in the endothelium of conduit arteries [54]. Yet, in mice overexpressing human IGF-1R in the endothelium insulin sensitivity is unaffected [55] To better understand the effects of IGF-1/IGF1R signaling on endothelial phenotype, subsequent studies should investigate transcriptional changes in the cerebromicrovascular endothelial cells derived from VE-cadherin-CreERT2/Igf1rf/f mice. While decreasing IGF-1 input to the endothelial cells is clearly detrimental, mice overexpressing human IGF-1R in the endothelium were shown to exhibit unaltered vasorelaxation to endothelium-dependent vasodilators [55].

The aforementioned observations are consistent with the concept that disruption of IGF-1/IGF1R signaling in endothelial cells promotes the acquisition of an accelerated neurovascular aging phenotypes. Accordingly, aging in humans and in laboratory animals results in circulating IGF-1 deficiency [12, 30, 32,33,34], which associates with neurovascular uncoupling, endothelial dysfunction, microvascular rarefaction and disruption of the blood–brain barrier [41, 56,57,58]. Heterochronic parabiosis is a surgical approach for joining the circulatory systems of an aged and a young animal that is used to identify non-cell autonomous mechanisms of aging. We have recently demonstrated that exposure to a young humoral environment rescues endothelial aging phenotypes in mice, including attenuation of oxidative stress and restoration of endothelium-mediated vasodilation [59]. Importantly, transcriptomic analysis identified IGF1R signaling as a likely upstream regulator involved in young blood-mediated vascular rejuvenation [59]. In future studies older VE-Cadherin-CreERT2/Igf1rf/f mice could be used as parabionts to experimentally interrogate the contribution of IGF-1/IGF1R signaling to the vasoprotective effects of young blood transfer.

Taken together, our present findings provide additional support for the concept that deficient IGF-1 input to the cerebromicrovascular endothelial cells compromises the function of the neurovascular unit, impairing NVC responses and likely multiple other aspects of brain health. The findings that disruption of IGF-1/IGF1R signaling results in neurovascular uncoupling and endothelial dysfunction have important translational relevance for the genesis of age-related vascular cognitive impairment and cognitive problems associated with genetic IGF-1 deficiency (e.g. in patients with growth hormone releasing hormone-receptor [GHRH-R] mutations, isolated GH deficiency or GH receptor gene defects [Laron syndrome]). Additionally, multiple IGF1R mutations have been described in children born small for gestational age (SGA) [60, 61], who later exhibit endothelial dysfunction [62] and have decreased levels of intelligence and various cognitive problems [63]. Future studies determining how IGF1R mutations in humans affect endothelial function and NVC responses as well as CBF should be quite revealing. The results of the present study, taken together with the findings of earlier investigations [9, 12, 24,25,26, 53, 64, 65], point to potential multifaceted benefits of various pharmacological, dietary [66, 66] and lifestyle interventions rescuing IGF-1 input to the cerebral microcirculation and the aging brain.