Early elevations in arterial pressure: a contributor to rapid depressive symptom emergence in female Zucker rats with metabolic disease?

Abstract

One of the growing challenges to public health and clinical outcomes is the emergence of cognitive impairments, particularly depressive symptom severity, because of chronic elevations in metabolic disease and cerebrovascular disease risk. To more clearly delineate these relationships and to assess the potential for sexual dimorphism, we used lean (LZR) and obese Zucker rats (OZR) of increasing age to determine relationships between internal carotid artery (ICA) hemodynamics, cerebral vasculopathies, and the emergence of depressive symptoms. Male OZR exhibited progressive elevations in perfusion pressure within the ICA, which were paralleled by endothelial dysfunction, increased cerebral arterial myogenic activation, and reduced cerebral cortex microvessel density. In contrast, female OZR exhibited a greater degree of ICA hypertension than male OZR but maintained normal endothelial function, myogenic activation, and microvessel density to an older age range than did males. Although both male and female OZR exhibited significant and progressive elevations in depressive symptom severity, these were significantly worse in females. Finally, plasma cortisol concentration was elevated higher and at a younger age in female OZR as compared with males, and this difference was maintained to final animal usage at ∼17 wk of age. These results suggest that an increased severity of blood pressure waves may penetrate the cerebral circulation more deeply in female OZR than in males, which may predispose the females to a more severe emergence of depressive symptoms with chronic metabolic disease, whereas males may be more predisposed to more direct cerebral vasculopathies (e.g., stroke, transient ischemic attack).

NEW & NOTEWORTHY We provide novel insight that the superior maintenance of cerebrovascular endothelial function in female versus male rats with chronic metabolic disease buffers myogenic activation of cerebral resistance arteries/arterioles despite worsening hypertension. As hypertension development is earlier and more severe in females, potentially due to an elevated stress response, the blunted myogenic activation allows greater arterial pressure wave penetrance into the cerebral microcirculation and is associated with accelerated emergence/severity of depressive symptoms in obese female rats.

INTRODUCTION

It has been well established that both the incidence and prevalence of chronic metabolic disease and disease risk continue to rise in developed economies worldwide (1, 2). Although this growth in obesity, impaired glycemic control, dyslipidemia, and hypertension represents risk factors for poor health outcomes spanning an array of disease conditions, a particularly challenging one for public health is the increased likelihood of developing significant cerebrovascular dysfunction, resulting in perfusion-based pathologies in the brain such as small vessel disease, stroke, transient ischemic attack, and a growing awareness of linkages to many cognitive impairments (3–5). Many of these elevated risk states associated with growing vasculopathies are associated with altered flow control to, and within, the brain. As these risk states can result in altered hemodynamic stresses such as pressure waveforms penetrating deeper into the cerebral microcirculation disrupting flow conditions (6, 7), a greater understanding of how these influences interact to produce poor health outcome is critical. To address these growing challenges to morbidity and mortality, as well as the economic costs that must be borne by society (3), it is imperative to gain an integrated understanding of the implications of evolving risk factors and the emerging cerebral vasculopathy within the context of an altered hemodynamic environment within the cerebral circulation.

The obese Zucker rat (OZR) is a useful model of chronic, evolving metabolic disease in humans. Although it manifests obesity as an outcome of severe leptin resistance, OZR develop the growing impairments to glycemic control, dyslipidemia, and moderate hypertension in a manner that is comparable to that determined in many humans because of the chronic hyperphagia (8–10). Furthermore, OZR demonstrate a sexual dimorphism that also mimics that determined in human subjects, wherein males manifest a severity of vascular dysfunction that is both widespread and more severe at earlier ages than that determined in females (11, 12). However, following early ovariectomy, female OZR lose this potential source for vascular protection under conditions of metabolic disease and manifest vascular outcomes that are indistinguishable from those in males (12). Although initial studies have investigated sex-based differences between male and female OZR and other models of metabolic disease (13–15) in terms of cerebral vasculopathy, it is important to extend observations into a more functionally applicable framework. As the ability to maintain protective cerebral autoregulatory mechanisms in the face of elevated perfusion pressures, and other components of metabolic disease, represents a critical aspect of maintaining optimal brain health, identifying relationships between perfusion pressures, cerebral vascular function, and the emergence of impaired cognitive outcomes represents a critical area of investigation.

The present study was designed to determine relationships between internal carotid artery (ICA) perfusion pressure with increasing age and cerebral vascular structure and function across the temporal window when vascular disease risk develops most rapidly within the OZR model of metabolic disease in both male and female animals. This study evaluated the hypothesis that cerebral resistance vessel structure and function were coupled to changes in ICA perfusion pressure to serve as an effective protective mechanism for the distal cerebrovascular microcirculation to minimize the risk for poor health and cognitive outcomes.

MATERIALS AND METHODS

Animals

Male and female LZR and OZR were purchased at ∼7 wk of age (Envigo) and were maintained on standard chow and drinking water ad libitum for the duration of the study. All animals were housed in an accredited animal care facility at either the University of Western Ontario or the West Virginia University Health Sciences Center, and all procedures had received prior Institutional Animal Care and Use Committee (IACUC) approval. Rats were utilized for terminal experiments at 8–9, 13–15, and 17–20 wk of age. The age ranges were selected as they represent key points in the development of metabolic and cerebrovascular disease risk as well as depressive symptom emergence from our previous studies in Zucker rats (16, 17). At the young age (8–9 wk), the worsening metabolic disease is evident, and there are indices of vasculopathy evident with some depressive symptom emergence. At the intermediate age (13–15 wk), metabolic disease is established, with more evident vasculopathy and the clear presence of depressive symptoms. Finally, at the adult age (17–20 wk), all three conditions are clearly manifest in the Zucker rat model. It should be noted that evolution of vasculopathy in female Zucker rats is delayed in comparison with age-matched males (11).

For this study, there were two major cohorts of rats used, the first for in vivo procedures and data collection relevant to perfusion pressures and hemodynamic conditions within the internal carotid artery of rats, and the second for ex vivo procedures and data collection relevant for higher-resolution vascular phenotypes to provide additional insight and context for results from the first cohort.

In Vivo Hemodynamic Experiments

In the first cohort of rats, animals were anesthetized in a plexiglass induction chamber using 4% vol/vol of isoflurane in oxygen using a flow rate of 1–1.2 L/min. Subsequently, animals received an endotracheal intubation and were connected to a control mechanical ventilator (CMV; SAR 1000, CWE; Geneq, Montreal, QC, Canada), set to a volume-controlled mode based on the individual animal body mass. All values for respiration rate (RR) and tidal volume (Vt) were based on reference values (18). To minimize any impact of isoflurane as a respiratory depressant, the postinduction isoflurane anesthesia dose was decreased to 2% with an oxygen flow of 1.0 L/min, and the animal was placed on a warming pad in a supine position. Finally, breathing pattern, RR, and the color of mucous membranes were monitored to ensure adequate ventilation, and the volume ventilation was set to I:E ratio of 30/70, and positive end-expiratory pressure was set to 3–6 mL H₂O. Body temperature was maintained at 36–38°C through a water-circulated heat pump (Gaymar, T/Pump; Braintree Scientific, Inc., Braintree, MA).

Prior to surgery, the solid-state pressure sensor(s), located on the tip of the 1.6-F pressure catheter (Transonic Scisense Inc., London, ON) was soaked in 0.9% saline for ∼20 min and then balanced to 0 mmHg against atmospheric pressure. Then the catheter was connected to amplifier ADV500 (Transonic Scisense Inc., London, ON, Canada) and to LabChart 8 data acquisition software, and analysis software (ADInstruments, Colorado Springs, CO) was used to collect pressure data.

Following anesthesia, the left carotid artery (LCA) was cleared of surrounding tissue via blunt dissection. Under stereomicroscopy, the LCA was separated from adjacent tissues and adventitia, and 5-0 sutures were placed around the LCA to be used for retraction and/or clamping and hemostasis. Following isolation, the LCA was briefly occluded using a 3-V microvascular clamp, and an incision was made in the free segment of the LCA, and the catheter was carefully inserted to avoid damage to either the catheter tubing shaft or sensor electrodes. Following placement, the catheter was secured using the 5-0 sutures.

As a final surgical step, the femoral vein was cannulated to facilitate infusion of pharmacological challenges (below) or supplemental saline, if needed. At this point, all surgical sites were covered in warmed physiological salt solution (PSS)-soaked gauze to minimize any evaporative water loss from those locations.

At the conclusion of the surgical procedures described earlier, and a 30-min period of recovery and equilibration for the anesthetized rat, perfusion pressure waveforms were collected via the arterial catheter for the subsequent 30 min using LabChart 8 data acquisition software.

Ex Vivo Vascular Structure and Function Experiments

All rats in this cohort were anesthetized with injections of sodium pentobarbital (50 mg·kg⁻¹ ip) and a carotid artery was cannulated for determination of mean arterial pressure. From each animal, a venous blood sample was also acquired (venipuncture) for the determination of circulating endocrine, oxidant, and inflammatory biomarkers. Sex hormone profiles were determined on a fee-for-service basis by a professional clinical laboratory using commercially available ELISA kits (Cayman, Mybiosource). Prior to the next step in the procedures, rats received a low dose of heparin (100 IU/kg iv) to prevent the formation of any blood clots during tissue harvest.

Investigation of Isolated Vessels

Following the initial surgery, each rat was decapitated, and the brain was removed from the skull case and placed in a cold physiological salt solution (PSS; 4°C). Subsequently, a middle cerebral artery (MCA) was dissected from its origin at the Circle of Willis, as described previously (19). Arteries were placed in a heated chamber (37°C) that allowed the lumen and exterior of the vessel to be perfused and superfused, respectively, with PSS equilibrated with 21% O₂, 5% CO₂, and 74% N₂ from separate reservoirs. Vessels were cannulated at both ends with glass micropipettes and were tied (10-0 nylon suture) to the inflow and outflow pipettes, which were connected to a reservoir perfusion system that allowed intraluminal pressure and gas concentrations to be controlled. Any side branches were ligated using a single strand teased from a 6-0 suture. Vessel diameter was measured using television microscopy and an on-screen video micrometer. Arteries were extended to their in situ length and were equilibrated at 80% of the animal’s mean arterial pressure at that age.

Active tone for pressurized MCA in the present study, calculated as (ΔD/D_max)·100, where ΔD is the diameter increase as measured at equilibration pressure while in normal PSS (with Ca²⁺) to that in Ca²⁺-free PSS, and D_max is the maximum diameter measured at the equilibration pressure in Ca²⁺-free PSS; these data are presented in Tables 1–3. Any vessel that did not demonstrate significant active tone at the equilibration pressure, defined as >25%, was discarded. Following equilibration, the dilator reactivity of MCA was assessed in response to increasing concentrations of acetylcholine (10⁻¹⁰ M–10⁻⁶ M) and in response to imposition of hypoxia. For the purposes of the present study, hypoxia was defined as a change in Po₂ of the superfusate/perfusate PSS from ∼135 mmHg (21% O₂, 5% CO₂, balance N₂ in the equilibration gas) to ∼45 mmHg (0% O₂, 5% CO₂, balance N₂ in the equilibration gas). These oxygen pressures have been previously validated with O₂ microelectrodes in the vessel chamber (19). After responses to acetylcholine and hypoxia were determined under control conditions, vessels were incubated with either L-NAME (NG-nitro-l-arginine methyl ester, 10⁻⁴ M) and/or indomethacin (10⁻⁵ M) for 45–60 min to assess the contributions from endothelial production of nitric oxide (16) or vasoactive metabolites via cyclooxygenase (i.e., prostacyclin (PGI₂) or thromboxane A₂ (TxA₂) (17).

Table 1.

Baseline characteristics of all animals within the youngest age group in the present study (8–9 wk)

	Male LZR	Female LZR	Male OZR	Female OZR
Mass, g	140 ± 5	120 ± 5*	175 ± 6*†	150 ± 5*†‡
[Glucose]blood, mg/dL	84 ± 4	82 ± 3	105 ± 6*†	106 ± 5*†
[Insulin]plasma, ng/mL	0.3 ± 0.1	0.3 ± 0.1	1.4 ± 0.3*†	1.2 ± 0.3*†
[TNF-α]plasma, ng/mL	0.9 ± 0.2	1.1 ± 0.2	2.5 ± 0.4*†	2.4 ± 0.4*†
[Nitrotyrosine]plasma, ng/mL	12 ± 3	12 ± 2	21 ± 5*†	20 ± 4*†
[Testosterone]plasma, pg/mL	2.8 ± 0.5	0.2 ± 0.1*	2.5 ± 0.4	0.3 ± 0.2*‡
[Estradiol]plasma, pg/mL	20 ± 4	48 ± 5*	18 ± 3	55 ± 6*‡
MCA active tone, %	41 ± 3	40 ± 3	39 ± 4	41 ± 3

n = 5 animals for each group in Cohort 1; n = 6 animals in each group for Cohort 2. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

*P < 0.05 vs. male LZR in the age group.

†P < 0.05 vs. LZR of that sex.

‡P < 0.05 vs. male OZR in the age group.

Table 3.

Baseline characteristics of all animals within the oldest age group in the present study (17–20 wk)

	Male LZR	Female LZR	Male OZR	Female OZR
Mass, g	377 ± 10	288 ± 9*	698 ± 14*†	522 ± 12*†‡
[Glucose]blood, mg/dL	94 ± 5	96 ± 4	180 ± 14*†	176 ± 15*†
[Insulin]plasma, ng/mL	0.5 ± 0.2	0.5 ± 0.2	7.7 ± 1.1*†	7.6 ± 0.9*†
[TNF-α]plasma, ng/mL	1.2 ± 0.3	2.2 ± 0.3*	7.5 ± 1.2*†	6.9 ± 1.0*†
[Nitrotyrosine]plasma, ng/mL	18 ± 4	17 ± 4	55 ± 6*†	49 ± 5*†
[Testosterone]plasma, pg/mL	3.2 ± 0.4	0.4 ± 0.2*	3.5 ± 0.4	0.5 ± 0.2*‡
[Estradiol]plasma, pg/mL	20 ± 4	67 ± 7*	33 ± 4*	74 ± 6*‡
MCA active tone, %	43 ± 3	42 ± 3	44 ± 2	41 ± 3

n = 5 animals for each group in Cohort 1; n = 6 animals in each group for Cohort 2. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

*P < 0.05 vs. male LZR in the age group.

†P < 0.05 vs. LZR of that sex.

‡P < 0.05 vs. male OZR in the age group.

Table 2.

Baseline characteristics of all animals within the middle age group in the present study (13–15 wk)

	Male LZR	Female LZR	Male OZR	Female OZR
Mass, g	312 ± 9	240 ± 8*	492 ± 12*†	382 ± 11*†‡
[Glucose]blood, mg/dL	89 ± 4	91 ± 3	158 ± 10*†	146 ± 11*†
[Insulin]plasma, ng/mL	0.4 ± 0.2	0.3 ± 0.1	4.5 ± 0.6*†	4.2 ± 0.4*†
[TNF-α]plasma, ng/mL	1.0 ± 0.2	1.6 ± 0.3	4.5 ± 1.1*†	5.4 ± 1.2*†
[Nitrotyrosine]plasma, ng/mL	16 ± 4	15 ± 3	35 ± 6*†	33 ± 5*†
[Testosterone]plasma, pg/mL	3.4 ± 0.5	0.3 ± 0.1*	2.9 ± 0.4	0.4 ± 0.2*‡
[Estradiol]plasma, pg/mL	19 ± 3	58 ± 6*	26 ± 4	67 ± 7*‡
MCA active tone, %	42 ± 2	43 ± 3	44 ± 3	42 ± 3

n = 5 animals for each group in Cohort 1; n = 6 animals in each group for Cohort 2. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

*P < 0.05 vs. male LZR in the age group.

†P < 0.05 vs. LZR of that sex.

‡P < 0.05 vs. male OZR in the age group.

Myogenic properties were assessed in MCA over the range of 20–160 mmHg, in randomized 20 mmHg increments. Pressure was changed nonsequentially, and vessels were allowed 10 min to equilibrate at each pressure before arterial inner and outer diameters were recorded. Following determination of myogenic activation under untreated control conditions, the endothelium of the MCA was removed by air bolus embolization, with the effectiveness of the procedure verified by loss of all vascular reactivity following challenge with 10⁻⁶ M acetylcholine (20).

Following the experimental procedures for measuring ex vivo vascular reactivity, the perfusate and superfusate PSS were replaced with Ca²⁺-free PSS containing the metal ion chelators EDTA (0.03 mM) and EGTA (2.0 mM). Vessels were challenged with serotonin (5-hydroxytryptamine; 10⁻⁶ M) until all active tone was lost. Subsequently, intralumenal pressure within the isolated vessel was altered, in 20 mmHg increments, between 0 and 160 mmHg. To ensure that a negative intralumenal pressure was not exerted on the vessel, 5 mmHg was used as the “0 mmHg” intralumenal pressure point; all other values of intralumenal pressure were multiples of 20 mmHg up to 160 mmHg. At each intralumenal pressure, the inner and outer diameters of the passive MCA were determined after 5–7 min of equilibration time (21).

Determination of Vascular Metabolites of Arachidonic Acid

Vascular production of 6-keto-prostaglandin F_1α (6-keto-PGF_1α; the stable breakdown product of PGI₂) (22, 23) and 11-dehydro-thromboxane B₂ (11-dehydro-TxB₂; the stable plasma breakdown product of TxA₂) (24), in response to challenge with hypoxia using pooled arteries (femoral, carotid, Circle of Willis, anterior/posterior cerebral arteries) from each rat, was measured using previously described techniques (25). Briefly, vessels were suspended in 1 mL of PSS within small glass test tubes and equilibrated gas containing 21% O₂, 5% CO₂, and balance N₂. After 30 min, the PSS was collected for analysis and replaced with fresh PSS, and the content of the equilibration gas was switched to 0% O₂, 5% CO₂, and balance N₂ for the subsequent 30 min. After that time the supernatant was collected and frozen in liquid N₂ until final analysis. Metabolite release by the vessels was determined using commercially available EIA kits for 6-keto-PGF_1α and 11-dehydro-TxB₂.

Determination of Microvessel Density

Following removal of the MCAs from the Circle of Willis on the base of the brain, the brain was placed within Tissue-Tek OCT compound and frozen. Brains were then sliced into 5 µm cross sections and were then stained using the established approach developed by Munzenmaier and Greene (26) using primary anti-CD-31 antibody. Under microscopy, localization of labeled microvessels was performed with a Nikon E600 upright microscope with a ×20 objective lens. The microscope was coupled to a cooled CCD camera (Micromax; Princeton Instruments Inc., Trenton, NJ). Five nearby 1 mm² images were taken from each of three sections in the frontal cortex of each brain, and the mean microvessel density (MVD) within these 15 images was taken to represent cortical MVD in that animal (26). All acquired images from individual sections were analyzed for number of microvessels using Nikon Elements software.

Mathematical Analyses

Mechanical responses following challenge with logarithmically increasing dosages of acetylcholine were fit with the logistic equation:

$$y=\min \hspace{0.17em}+\left[\frac{\max \hspace{0.17em}-\min \hspace{0.17em}}{1+{10}^{\log \hspace{0.17em}E{D}_{{50}^{-x}}}}\right],$$

where y represents the vessel diameter; “min” and “max” represent the lower (minimum) and upper (maximum) bounds, respectively, of the change in diameter with agonist concentration; x is the logarithm of the agonist concentration; and logED₅₀ represents the logarithm of the agonist concentration (x) where the response (y) is halfway between the bounds. For the presentation of results, we have focused on the changes in the upper bounds as a representation of vessel reactivity, as the lower bound remained consistent between all groups (defined as the prechallenge diameter), and we did not determine a consistent or significant change to the logED₅₀ values between treatment groups. As a result of this approach, the upper bound represents that statistically determined asymptote for the concentration-response relationship and does not assume that the vascular response at the highest utilized concentration of the agonist represents the maximum possible response. Rather, the sigmodal relationship of best fit to the data will predict the statistical upper bound of the response given the data points entered into the model. As such, the upper bound is frequently slightly larger than the dilator response of the vessel at the highest concentration of the agonist.

The myogenic properties of MCA from each experimental group were plotted as mean diameter at each intraluminal pressure and fitted with a linear regression (y = α₀ + βx), where the slope coefficient β represents the degree of myogenic activation (δ diameter/δ pressure). Increasingly negative values of β therefore represent a greater degree of myogenic activation in response to changes in intralumenal pressure.

All calculations of passive arteriolar wall mechanics (used as indicators of structural alterations to the individual microvessel) are based on those used previously (27) with minor modification. Vessel wall thickness was calculated as follows:

$$\mathrm{W}\mathrm{T}=\frac{(\mathrm{O}\mathrm{D}-\mathrm{I}\mathrm{D})}{2},$$

where WT represents wall thickness (µm), and OD and ID represent arteriolar outer and inner diameter, respectively (µm).

For the calculation of circumferential stress, intralumenal pressure was converted from mmHg to N/m², where 1 mmHg = 1.334 × 10² N·m². Circumferential stress (σ) was then calculated as follows:

$$\frac{\mathrm{\sigma }=(P_{\mathrm{I}\mathrm{L}}\times \mathrm{I}\mathrm{D})}{\left(2\mathrm{W}\mathrm{T}\right)}.$$

Circumferential strain (ε) was calculated as follows:

$$\mathrm{\epsilon }=\frac{(\mathrm{I}\mathrm{D}-\mathrm{I}{\mathrm{D}}_5)}{\mathrm{I}{\mathrm{D}}_5},$$

where ID₅ represents the internal arteriolar diameter at the lowest intralumenal pressure (i.e., 5 mmHg). The stress versus strain relationship from each vessel was fit (ordinary least squares analyses, r² > 0.85) with the following exponential equation:

$$\mathrm{\sigma }={\mathrm{\sigma }}_5{\mathrm{e}}^{\mathrm{\beta }\mathrm{\epsilon }},$$

where σ₅ represents circumferential stress at ID₅ and β is the slope coefficient describing arterial stiffness. Higher levels of β are indicative of increasing arterial stiffness (i.e., requiring a greater degree of distending pressure to achieve a given level of wall deformation).

Behavioral Characteristics: Coat Status

The coat status evaluation (28) was done within 48 h of the final usage of any animal in the present study. The total cumulative score was computed by giving an individual score of 0 (clean) or 1 (dirty) to eight body parts (head, neck, dorsal coat, ventral coat, tail, forelimb, hindlimb, and genital region).

Sucrose Spray Test

The sucrose spray test (28) was used to evaluate acute grooming behavior, defined as cleaning of the fur by licking or scratching. A 10% sucrose solution was sprayed on the dorsal coat of each rat, and grooming activity was recorded for 5 min. The viscosity of the sucrose solution will dirty the coat and induce grooming behavior, with depressive symptoms characterized by an increased latency (idle time between spray and initiation of grooming) and decreased frequency (number of times grooming a particular body part).

Novelty Suppressed Feeding Test

Within 48 h of final usage, all access to food was removed from the rats for 24 h (ad libitum access to water was continued) (29). Subsequently, individual rats were placed in one corner of an empty cage (18 in. × 24 in.) with fresh bedding. One pellet of the normal chow was placed in the center of the cage. The time taken from placement of the food pellet to the moment when the rat began consuming the food pellet (i.e., not just sniffing and handling the pellet). At this time, the animal was removed from the cage and returned to its home cage with normal access to food restored until final animal usage.

Full presentations and discussions about the above-utilized determinations of depressive symptom severity are summarized in Refs. 28–30.

Statistical Analyses

All data in the tables are presented as means ± SE. All data in the figures are presented as raw data from each experiment and as “box and whisker” plots. The lower boundary of the box indicates the 25th percentile, the line within the box indicates the median, and the upper boundary of the box indicates the 75th percentile. The “whiskers” (error bars) above and below the box indicate the 90th and 10th percentiles, respectively. Differences in all calculated parameters or descriptive characteristics between the different experimental groups in the present study were assessed using analysis of variance (ANOVA). A one-way ANOVA was used to assess differences based on sex, with a separate ANOVA used to assess differences based on metabolic disease. The Student–Newman–Keuls (SNK) test was used as the post hoc test for differences in means. The SNK test was selected given its general neutrality and balance between power and the probability of making a type 1 error as compared with other post hoc tests. In all cases, P < 0.05 was taken to reflect statistical significance.

RESULTS

Results from the Determination of In Vivo Hemodynamics

Data describing the pressures within the ICA in male and female OZR over the age ranges in the present study are summarized in Fig. 1. In the youngest age cohort of animals (Fig. 1A), female LZR consistently exhibited an increased systolic, diastolic, and mean arterial pressure as compared with male LZR. In both sexes, all three ICA pressures were also elevated by the growing metabolic disease in OZR as compared with their LZR controls. Finally, there was evidence that the increased pressures (SBP, DBP, and mean) were increased to a greater extent in female OZR as compared with males at 8–9 wk of age. At 13–15 wk of age (Fig. 1B), the increases in systolic, diastolic, and mean arterial pressures with growing severity of the metabolic disease were somewhat greater than at the younger age in OZR, with the disparity between male and female OZR becoming somewhat larger. However, differences in the three pressure measurements between male and female LZR were no longer present. In the oldest cohort of animals (17–20 wk), any differences in the three pressures measured between the sexes were largely absent, and all differences largely reflected the impact of the metabolic disease itself (Fig. 1C). Representative traces of ICA pressure for animals within each of the experimental groups within the present study are presented in Fig. 2.

Figure 1.

Internal carotid artery blood pressures in male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for rats at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age and are presented for systolic blood pressure, diastolic blood pressure, and mean arterial pressure. n = 5 or 6 in each animal group. *P < 0.05 vs. LZR for that sex, †P < 0.05 vs. males within that strain. Please see text for additional details. LZR, lean Zucker rats; OZR, obese Zucker rats.

Figure 2.

Representative traces for internal carotid artery (ICA) pressure from individual animals within each of the experimental groups within the present study. Data are collected at 100 Hz and presented for 24 s of recording in 8- to 9-wk-old (Young) LZR and OZR males (A) and females (B), 13- to 15-wk-old (Intermediate) LZR and OZR males (C) and females (D), and 19- to 20-wk-old (Adult) LZR and OZR males (E) and females (F). In all panels, male LZR are black, male OZR are red, female LZR are blue, and female OZR are green. Please see text for additional details. LZR, lean Zucker rats; OZR, obese Zucker rats.

Results from Ex Vivo Vascular Procedures

Figure 3 presents the changes in cerebral cortex microvessel density in male and female LZR and OZR at increasing ages/severity of metabolic disease. At the youngest age range, there were no differences in MVD between male LZR, female LZR, and female OZR, although this was reduced in male OZR (Fig. 3A). At the intermediate age range, this pattern was replicated, although a trend toward reduced MVD was evident in female OZR as compared with female LZR (Fig. 3B). At the oldest age group, while cerebral MVD was comparable between male and female LZR, a rarefaction in the brains of OZR had developed to the point where MVD was reduced in both sexes as compared with their respective LZR control (Fig. 3C).

Figure 3.

Cerebral cortical microvessel density in male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for rats at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age. n = 8 in each animal group. *P < 0.05 vs. LZR for that sex, †P < 0.05 vs. males within that strain. Please see text for additional details. LZR, lean Zucker rats; OZR, obese Zucker rats.

The changes in wall stiffness of isolated MCA from male and female LZR and OZR in the present study are summarized in Fig. 4. At the youngest age range, there were no differences between the slope coefficient (β) of the stress versus strain relationship between any of the four groups (Fig. 4A). At the intermediate age range, β tended to be increased in both male and female OZR as compared with LZR, although this was sufficiently variable that it did not reach statistical significance (Fig. 4B). At the oldest age range, the slope coefficient describing the stress versus strain relationship for MCA in male and female OZR was significantly elevated as compared with that in the respective control LZRs but were not significantly different from each other (Fig. 4C).

Figure 4.

The slope (β) coefficient for the circumferential stress vs. strain relationship of the wall of ex vivo MCA from male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for rats at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age. n = 8 in each animal group. *P < 0.05 vs. LZR for that sex. Please see text for additional details. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

The dilator responses of MCA in response to challenge with hypoxia are summarized in Fig. 5. At the young (Fig. 5A), intermediate (Fig. 5B), and oldest (Fig. 5C) age ranges, both male and female LZR demonstrated robust responses to reduced Po₂, with no significant differences determined at any time point. However, at the young age range, MCA from male OZR demonstrated a reduced hypoxic dilation as compared with that in LZR, whereas the responses in MCA from female OZR remained intact (Fig. 5A). The impaired dilator response to hypoxia in MCA from male OZR was increased at the intermediate age range, whereas the responses in vessels from female OZR remained largely normal (Fig. 5B). At the older age range, dilator responses of MCA from both male and female OZR were significantly reduced as compared with that in their respective control LZR (Fig. 5C).

Figure 5.

Hypoxic dilation of ex vivo MCA from male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for rats at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age. n = 8 in each animal group. *P < 0.05 vs. LZR for that sex. Please see text for additional details. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

The impact of inhibitors of NOS (L-NAME) and cyclooxygenase (COX, with indomethacin) on dilator responses to hypoxia is summarized in Fig. 6. In LZR of both sexes, regardless of age, hypoxic dilation of MCA was a function of the combined impact of both nitric oxide production and the production of metabolites from COX, most likely PGI₂ (Fig. 6, A–C). With the growing progression of metabolic disease in OZR, the nitric oxide component of the dilator response to hypoxia was reduced in both male and female OZR, whereas the net effect of COX inhibition was increasingly ineffective with age (Fig. 6, D–F).

Figure 6.

The impact of pharmacological treatment of ex vivo MCA with L-NAME, indomethacin, or both, on hypoxic dilation in male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for LZR at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age and for OZR at 8–9 (D), 13–15 (E), and 19–20 (F) wk of age. n = 8 in each animal group. *P < 0.05 vs. responses in untreated vessels in that sex or strain. Please see text for additional details. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

Figure 7 summarizes the dilator responses of MCA from male and female LZR and OZR of increasing ages in response to acetylcholine. The responses determined were comparable with those for challenge with hypoxia, with an increasingly attenuated response determined in MCA from male OZR with increasing age and severity of metabolic disease (Fig. 7, A–C). In contrast, dilation of MCA from female OZR was comparable with that from vessels in female LZR at the young (Fig. 7A) and intermediate (Fig. 7B) age range, with a reduced reactivity demonstrated only at the older age range (Fig. 7C).

Figure 7.

Acetylcholine-induced dilation of ex vivo MCA from male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for rats at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age. n = 8 in each animal group. *P < 0.05 vs. LZR for that sex. Please see text for additional details. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

Figure 8 presents the impact of treatment of ex vivo MCAs with L-NAME and/or indomethacin on dilator responses to acetylcholine. In LZR of both sexes, regardless of age, hypoxic dilation of MCA was largely a function of nitric oxide production, with a very modest contribution from COX metabolites (Fig. 8, A–C). As the severity of metabolic disease worsened in OZR, the loss of dilator responses to acetylcholine in MCA from both males and females was largely reflective of a loss of nitric oxide bioavailability as the impact of L-NAME treatment was increasingly blunted. The impact of COX inhibition via indomethacin remained without significant impact (Fig. 8, D–F).

Figure 8.

The impact of pharmacological treatment of ex vivo MCA with L-NAME, indomethacin, or both, on acetylcholine-induced dilation in male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for LZR at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age and for OZR at 8–9 (D), 13–15 (E), and 19–20 (F) wk of age. n = 8 in each animal group. *P < 0.05 vs. responses in untreated vessels in that sex or strain. Please see text for additional details. LZR, lean Zucker rats; MCA, middle cerebral artery; OZR, obese Zucker rats.

The production of vasoactive metabolites from pooled arteries in response to challenge with hypoxia is presented in Fig. 9. Vascular production of TxA₂, estimated through production of 11-dehydro-TxB₂, was elevated in OZR males versus LZR males in all age cohorts (Fig. 9, A–C), with the difference increasing with age and the duration/severity of metabolic disease. Vascular production of TxA₂ in female OZR was not different from that in female LZR at the young (Fig. 9A) and intermediate (Fig. 9B) age cohorts but was significantly increased in the adult cohort (Fig. 9C). However, at no time did TxA₂ production in arteries from female OZR reach the level determined in age-matched male OZR. In contrast, vascular production of PGI₂, estimated through production of 6-keto-PGF_1α, was progressively reduced in male OZR versus LZR with increasing age (Fig. 9, D and E), an effect that was not determined in female OZR until the adult cohort (Fig. 9F).

Figure 9.

Vascular production of TxA₂ (based on production of 11-dehydro-TxB₂; A–C) and PGI₂ (based on production of 6-keto-PGF_1α; D–F), in pooled arteries of male and female LZR and OZR of increasing ages. Data are summarized as box and whisker plots for vessels following 30-min exposure to hypoxia (0% O₂ in the equilibration gas for the pooled arteries). n = 8 in each animal group. *P < 0.05 vs. LZR for that sex, †P < 0.05 vs. male OZR. Please see text for additional details. LZR, lean Zucker rats; PGI₂, prostacyclin; Tx, thromboxane; OZR, obese Zucker rats.

The myogenic activation of MCA from male and female LZR and OZR in the present study is presented in Fig. 10. At the youngest age range, no significant differences were identified in myogenic activation between the four groups, although there was a trend toward increased pressure-induced constriction in the male OZR as compared with responses in male LZR (Fig. 10A). These responses were comparable in the intermediate age range as well, with the only difference being an increased slope of the myogenic activation in male OZR versus male LZR (Fig. 10B). At the oldest age range, myogenic activation of MCA in male OZR was significantly increased as compared with all other groups, whereas that in female OZR was increased as compared with responses in control LZR (Fig. 10C). In response to removal of the endothelium from MCA, the increased myogenic activation determined in vessels from male OZR was consistently reduced toward normal levels, although this restoration was limited. A comparable observation was also found in MCA from female OZR at the oldest age range, where myogenic activation was significantly increased.

Figure 10.

The slope (β) of myogenic activation of ex vivo MCA from male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for rats at 8–9 (A), 13–15 (B), and 19–20 (C) wk of age under both untreated control conditions and following endothelium denudation following air bolus embolization. n = 8 in each animal group. *P < 0.05 vs. LZR for that sex, †P < 0.05 vs. OZR within that sex. Please see text for additional details. LZR, lean Zucker rats; OZR, obese Zucker rats.

Results from Behavioral Assessments

Figure 11 presents the changes in coat score for male and female LZR and OZR across the ranges of the present study. Although LZR of both sexes maintained an excellent coat status and level of grooming, OZR of both sexes did not. Both male and female demonstrated a progressively deteriorating maintenance of coat status, with females exhibiting this depressive symptom more severely than males (Fig. 11A). The responses of rats to novelty suppressed feeding demonstrated that there was not an age-dependent effect in LZR of either sex, but that both male and female OZR exhibited a prolonged time to feeding with increased age and metabolic disease severity (Fig. 11B). Although there was a tendency for female OZR for a longer response time than male OZR, this was not statistically significant. In response to sucrose spray, the latency to begin grooming was significantly increased in both sexes of OZR in comparison with their respective LZR controls (Fig. 11C), whereas the frequency of grooming following spray was reduced (Fig. 11D). The increase in latency and decrease in frequency was greater in female OZR than in male OZR. Plasma cortisol levels were significantly increased in male and female OZR as compared with LZR, with levels of this hormone most elevated in females (Fig. 11E).

Figure 11.

Markers of depressive symptom severity in male and female LZR and OZR at increasing ages. Data are summarized as box and whisker plots for coat score (A), the time for food consumption (B), latency to groom following sucrose spray (C), frequency of grooming following sucrose spray (D), and plasma cortisol levels (E) for rats at 8–9 (Young), 13–15 (Intermediate), and 19–20 (Adult) wk of age. n = 8 in each animal group. *P < 0.05 vs. LZR of that sex at that age, †P < 0.05 vs. male OZR. Please see text for additional details. LZR, lean Zucker rats; OZR, obese Zucker rats.

DISCUSSION

The present study aimed to elucidate the relationships between perfusion pressures, cerebrovascular adaptations, and the emergence of depressive symptoms in male and female LZR (Lean Zucker rats) and OZR (Obese Zucker rats) as the severity of metabolic disease evolved. Overall, our results indicate that male OZR experience a gradual increase in perfusion pressure in the internal carotid artery across the age range of the study. However, this increase is accompanied by MCA (middle cerebral artery) myogenic activation and alterations in microvascular network density, which contribute to increased perfusion resistance and protect the cerebral microcirculatory environment. Over this age range, male OZR exhibit a progressive emergence of depressive symptoms of moderate severity. In contrast, female OZR show a more rapid and severe elevation in ICA (internal carotid artery) pressure during the development of metabolic disease. Despite this, they manifest more normal patterns of MCA reactivity for a longer duration. However, this prolonged exposure to higher perfusion pressures and pressure waves in the cerebral microcirculation is associated with a faster and more severe emergence of depressive symptoms.

The distinct alterations in cerebral vasculature between male and female OZR in response to the worsening severity of metabolic disease likely play a fundamental role in driving these differential outcomes. Microvessel density within the cerebral cortex was reduced further and more rapidly in male OZR than in female OZR (Fig. 3). This progressive rarefaction, while representing a relatively modest contributor to alterations in perfusion resistance (31), will more significantly impair tissue oxygenation in subregions within the cerebral microcirculation (17). Vascular wall mechanics, estimated via the stiffness of the MCA wall to increasing intralumenal pressure, did not demonstrate significant divergence between male and female OZR at increasing ages. The predictable increase in wall stiffness with increasing severity and duration of metabolic disease was present in OZR and was comparable between the two sexes. However, given the changes to both endothelial function and myogenic activation (discussed below), it is likely that the pressure waveforms in female OZR may penetrate further and with greater severity than that in males, potentially representing another significant contributor to the differential outcomes.

The differential penetrance of pressure waves into the brain between obese females and males is an intriguing area of study. These sex-specific variations in cerebrovascular responses could have significant implications for overall brain health and function. If pressure waves penetrate more deeply into the brain in obese females, it could affect blood flow dynamics, oxygen delivery, and waste removal (6, 32). Furthermore, a dysregulation of cerebral blood flow can contribute to conditions like ischemia or hyperperfusion, both of which can impact brain health. Moreover, increased pressure wave penetrance might lead to microvascular damage contributing to cognitive decline or neurological disorders (33). Chronic exposure to pressure waves could also trigger neuroinflammatory responses or disruption of blood-brain barrier (BBB) function (34). Inflammation in the brain is associated with various conditions, including neurodegenerative diseases (e.g., Alzheimer’s, Parkinson’s) (34), whereas sex-specific differences in inflammation or exposure to toxins after BBB may play a role in determining susceptibility to these diseases (35). Pressure waves could also directly impact neurons and their circuits as they rely on precise signaling and communication. Changes in pressure dynamics might disrupt synaptic transmission, affecting memory, cognition, and mood while influencing neurotransmitter systems involved in mood regulation (33).

Alterations to endothelial function between male and female OZR with evolution of metabolic disease exhibited a prolonged maintenance of dilator responses to hypoxia and challenge with acetylcholine in females that was not present in males. Although previous studies have provided evidence into this superior maintenance of vascular endothelial function in female OZR versus males (12), and in other models as well (15), the present results do provide additional insight into the temporal nature of these differences. In addition, the results of the present study also suggest that there is a prolonged maintenance of vascular NO bioavailability in female OZR in response to challenge with either hypoxia or acetylcholine. Although these results are consistent with recent work in conduit and resistance arteries of male and female LZR and OZR (11, 12), the present results also provide some support for altered responses in endothelial arachidonic acid metabolism with metabolic disease. In previous studies, dilator responses to hypoxia in OZR were impacted far less by inhibition of COX than would have been expected (36). Given that this response in MCA has been demonstrated to reflect NO bioavailability and the production/actions of PGI₂, blockade of COX with indomethacin should have resulted in a reduced hypoxic dilation of MCA from OZR. However, indomethacin is equally effective at inhibiting both major isoforms of cyclooxygenase, COX-1 and COX-2, which means that it is also very effective at inhibiting the production of both PGI₂ and TxA₂ via either enzyme (22). Based on additional results in the present study, which are consistent with previous efforts (36), the imposition of hypoxia to arteries from OZR results in a reduced production of PGI₂ and an increased generation of TxA₂. As blockade of COX with indomethacin will reduce the production of both the dilator (PGI₂) and the constrictor (TxA₂), the treatment may be without a net impact on the responses of MCA from OZR as metabolic disease worsens.

One of the major observations from the present study is the difference in myogenic activation of MCA from male and female OZR with increasing severity of metabolic disease. Not only did female OZR exhibit a greater level of hypertension in the ICA than determined in age-matched males, but the increases in the strength of myogenic activation identified in male OZR were not paralleled by similar changes in females (Fig. 10). With an increase in myogenic responses in female OZR delayed by multiple weeks, this should contribute to an increased exposure of the cerebral microcirculation to higher pressure waves than in control LZR or in male OZR, given the alterations to resistance arteriolar wall mechanics discussed earlier. Although the increase in MCA myogenic activation in males with increased hypertension has been well established (37), the present results suggest that there may be a significant difference in how the cerebral resistance vasculature of females responds to increasing pressure. The present results suggest that part of the difference in myogenic activation is related to maintained endothelial function in female OZR despite the greater chronic elevations in perfusion pressure. This may be explained through several contributors. First, the maintained presence of circulating sex hormones in female versus male OZR (Table 1) has been established to contribute to a greater maintenance of vascular endothelial function and NO bioavailability through a variety of signaling mechanisms (38, 39). In addition, a recent study has suggested that, under conditions of significant elevations in arterial pressure, the ultimate increase in myogenic activation acts to reduce wall stress in the vessel in question and may serve as a protective mechanism to help maintain overall endothelial function to an extent that would be greater than predicted based on the impact of more moderate elevations in pressure (37). Furthermore, previous work investigating the role of the endothelium in modulating myogenic activation suggests that the production of NO from the vascular endothelium can be scavenged by oxidant radicals such as superoxide (40), which may contribute to an increased myogenic activation based on the ability of the generated peroxynitrite to inhibit K_Ca channels on vascular smooth muscle, blunting membrane hyperpolarization in the face of elevated calcium (41). Taken together, it can be speculated that the prolonged maintenance of endothelial function in female OZR as compared with that in males may reflect the impact of circulating female sex hormones better maintaining endothelial function and NO bioavailability, a greater antioxidant and anti-inflammatory defense (38, 39). This could delay the overall production of oxidant radicals that can inhibit VSM hyperpolarization with elevated calcium, resulting in greater buffering of myogenic activation and a reduction in perfusion resistance upstream of the cerebral microcirculation. It is important to note that a recent study of myogenic activation of ex vivo MCA from male OZR with varied levels of hypertension indicated that the increase in arterial blood pressure was well correlated with any elevation in the myogenic response [i.e., the slope (β) of the response]. However, there was no evidence that the range of intralumenal pressures over which the myogenic response was active had been altered because of the chronic hypertension associated with the metabolic disease (37).

This is the first study to report the temporal development of hypertension in female OZR manifesting metabolic disease, with the somewhat unexpected observation that ICA pressure was elevated more quickly and more severely than that in age-matched males. Although sex-based differences in terms of blood pressure control have been demonstrated in terms of the sympathetic nervous system, the renin-angiotensin-aldosterone system, and immune responses (42–45), these appear to be more associated with shifts in signaling and weighted contributions rather than outright differences in terms of holistic inputs to pressure control (46). There is also a clear understanding that there is a superior maintenance of vascular endothelial function in females versus males, even with increased metabolic disease risk, that may be associated with sex hormone profiles (38). However, it is of note that the emergence of depressive symptoms in female OZR was both more rapid and more severe than that in males. Within these results, the levels of cortisol in female OZR, and in some cases within female LZR, were significantly elevated as compared with the age-matched males. Given the utility of circulating cortisol or corticosterone as a marker of chronic stress- and anxiety-related symptoms, this observation would suggest that female OZR (and LZR) experience higher levels of chronic stress and anxiety than do males, despite being housed within the same location and environment. As determined with other models of depressive symptom development (47, 48), this chronically increased cortisol in the face of elevated levels of stress and anxiety may contribute to not only the more rapid development of depressive symptoms but also a greater degree of hypertension at any specific age within the present study. Recent work by Alshammari (49) suggests that differences in function within the HPA axis of male and female rats under stressed conditions will be associated with elevated corticosterone levels in females, a potentially powerful contributor to the emergence of sex-based differences in the emergence and severity of depressive symptoms. Finally, although a recent scoping review identified clear elevations in stress- and anxiety-sensitivity in human subjects with elevated cardiovascular and metabolic disease risk (50), the current literature does not provide sufficient resolution to clearly identify or gain insight into the impact and importance of potential sexual dimorphisms within this elevated risk subgroup at this time.

When considering the results from the present study, we can speculate on the differences in cerebrovascular adaptation to metabolic disease and the resulting health outcomes between male and female OZR. In males, the growing metabolic disease, including hypertension, is closely matched by vascular mechanisms that increase resistance to perfusion into the cerebral microcirculation. This limits the penetrance of pressure waves into the deeper microvessels and capillaries. However, this adaptation is associated with greater endothelial dysfunction, reducing vascular nitric oxide (NO) bioavailability, and an unfavorable ratio of endothelial-derived production of PGI₂ to TxA₂. In contrast, female OZR exhibit a higher degree of hypertension in the ICA at a younger age, possibly due to increased stress and anxiety. Despite this, females maintain superior vascular endothelial function, which buffers against expected shifts in myogenic activation in cerebral resistance vessels. As a result, stronger pressure waves can penetrate deeper into the microcirculation. Unfortunately, this condition is also associated with a more rapid and severe development of depressive symptoms in female OZR compared with males.

Speculatively, do these differences in the adaptation of the cerebral vasculature to a growing hypertensive and metabolic disease challenge, combined with a greater stress-sensitivity in female versus male Zucker rats, result in a condition wherein the predisposition to cerebral health outcomes is shifted between males and females? Are female OZR, with a healthier vascular endothelium, more likely to suffer from depressive symptoms at an earlier age than are males, owing to a greater penetrance of pressure waves into the deeper microcirculation? In contrast, are male OZR, with a more dysfunctional cerebral vascular endothelium but an increased myogenic activation, more protected from pressure wave penetrance and depressive symptom emergence, but at a greater risk for other cerebrovasculopathies, such as thromboemboli, transient ischemic attack, occlusive and hemorrhagic stroke, etc.? Is a concept of “pressure-dependent” versus “endothelium-dependent” shifts informative for considering the likelihood in cerebral and cerebrovascular outcomes between males and females afflicted with poorly controlled metabolic disease? Recent studies, while not providing information to directly address these questions, provide some insight into relationships that may be of relevance. Although it has been established that women are approximately twice as likely to be diagnosed with depression as men (51), one of the most common outcomes following stroke is “poststroke cognitive impairments” (PSCI). In 2023, a multicenter study demonstrated that PSCI are more common in woman (63%) than in men (39%) following stroke, although a differential impact on domains was identified with women experiencing greater impact on executive function and language and men experiencing deficits in verbal memory (52). In addition, Bako et al. (53) demonstrated that women experience greater PSCI than do men, especially in the early poststroke period, and hypothesized that female sex hormones, particularly progesterone, associated with increased expression and signaling of brain-derived neurotrophic factor following ischemic stroke, were predictive of improved cognitive outcomes. Other studies have suggested the importance of estradiol levels in contributing to sex differences in PSCI (54–56). Of direct relevance to the results of the present study, a recent review from Robison et al. (57) collates previous data suggesting that the presence of chronic metabolic disease in women results in a higher risk of cerebrovascular dysfunction resulting in both ischemic stroke and vascular dementia as compared with men. Based on the current results and these previous studies, it is possible that the cerebrovascular environment in females suffering from metabolic disease leads to a condition that predisposes the afflicted individual to cognitive impairments, which could be strongly exacerbated following stroke if they do not emerge prior to the stroke event.

The current results illustrate the importance of several areas for future investigation that were outside of the scope of the current study. To gain a more accurate and in-depth understanding of the role of cerebral vascular resistance in these relationships, it is imperative to gather high-resolution data on blood flow within the ICA and into the cerebral resistance vasculature. In addition, it is important for investigators to continue investigating the impact of male and female sex hormone profiles on integrated patterns of vascular reactivity (i.e., endothelial function and how that may impact myogenic activation in a hypertensive environment). It is also vital that these efforts not only determine mechanistic contributors to these outcomes, but that they also determine the temporal relevance and impacts as well, especially given that the levels of these hormone profiles can be extremely variable in time and with different elevated risk states (39). Finally, as the linkages between vasculopathy, impairments to perfusion/hemodynamic parameters, and the emergence of cognitive impairments and depressive symptoms continue to be revealed and appreciated, a higher-resolution understanding of these relationships and how they relate, as well as potential sites for intervention, appears to be well justified. This has been an area of increasing attention in recent years as numerous efforts have demonstrated the close linkages between indices of vascular dysfunction (including increased wall stiffness) and major cerebrovascular disease end points through excessive pressure pulsatility within the microcirculation (58). An ongoing study has also demonstrated relationships between chronic elevations in pressure pulsatility and the onset of cognitive impairments, specifically vascular dementia and Alzheimer’s disease (59). It is important to note that recent work has highlighted the importance of monitoring the impact of antihypertensive therapies, particularly for those impacting Ca²⁺-channels, the renin-angiotensin-aldosterone system, and mineral metabolism, on changes to arterial stiffness and pressure wave penetrance into the microcirculation and the resulting risk for end-organ damage (60).

van Sloten et al. (13) recently provided an overview of potential linkages between cerebral microvasculopathy and depression under conditions of metabolic disease, in this case type II diabetes mellitus. In their article, the authors link obesity, impaired glycemic control, and hypertension to many of the identical vasculopathies determined in this study (e.g., increased wall stiffness, impaired endothelial function, potential impairments to cerebral blood flow autoregulation) through altered neuronal metabolism and connectivity and, ultimately, the emergence of cognitive impairments and depression. Given the profound implications that this will have for public health and health policy in general, as well as the considerations that may be necessary to address sex-based differences in the progression, manifestation, and long-term outcomes of these integrated pathological conditions, ongoing investigation into these issues appears strongly warranted.

The issue of accelerated vascular/microvascular aging with elevated disease risk has gained increasing attention in recent years, and results from the present study may be relevant to these concepts. The evolution of metabolic disease in male OZR is comparable in many ways to accelerated vascular/microvascular aging in humans, with alterations in vascular wall mechanics, impaired endothelial function, and microvessel density all paralleling changes in humans with comparable risk factor profiles (8–10, 16). In female OZR, like that determined in female patients with metabolic disease, accelerated vascular aging is blunted while normal sex hormone levels are present but becomes virtually indistinguishable from that in males in their absence (11, 12). Excellent recent reviews on accelerated vascular/microvascular aging have been produced (e.g., Ref. 61), which implicate many conditions in the process that are highly comparable with those identified in the current study, including markers of elevated oxidant stress, vascular metabolite bioavailability, and chronic inflammation, among others. Notably, the ability to use multiscale study of translationally relevant models of human disease and disease risk to help guide insight into complex health outcomes, as discussed in van Sloten et al. (13), represents a vital avenue for future investigation.

The obese Zucker rat is a translationally relevant model for obesity and metabolic disease in humans, given its foundation in hyperphagia (9). Furthermore, as discussed earlier, there are clearly parallels in the linkages between metabolic disease, cerebral vasculopathy, and the emergence of depressive symptoms in human subjects afflicted with similar conditions—with the additional issue of sexual dimorphism to be considered. As evidenced in our recent study in male OZR, the presence of a worsening proinflammatory condition within the vasculature in parallel with declining glycemic control leads to a shift in arachidonic acid metabolism toward the increased production of TxA₂. This increased TxA₂ production is a significant predictor of cerebral microvascular rarefaction, with impacts on oxygenation of small regions within the cerebral cortex (17). Based on the present results, these effects—while apparently present in female OZR—are temporally discordant with the emergence of depressive symptoms in females and may reflect a contributor to a poor outcome rather than a causal mechanism. However, the early appearance of hypertension in the ICA of female rats (OZR > LZR), which may be associated with an enhanced stress/anxiety response, is more coupled to the temporal development of depressive symptom emergence than in males. Taken together, these data suggest that an aggressive prodromic effort at disease risk modification may be warranted in blunting the development of cognitive impairments with chronic metabolic disease. Although in males, aggressive focus on glycemic control and inflammatory status may result in delayed cerebral vasculopathy and depressive symptom emergence through blunting of TxA₂-initiated rarefaction, this may not be as effective or significant in females. Given the longer period of maintained vascular and endothelial function in females, it may be more beneficial to target blood pressure control much more aggressively. This can contribute to reducing alterations to vessel wall mechanics and pulse wave penetrance and may be most effective through antihypertensive therapies, or potentially ones designed to reduce stress and anxiety as a contributor to the elevated pressure (59, 60).

In summary, the results of the present study provide for multiple compelling avenues for future investigation. Although these results provide additional support for the impact of chronic, uncontrolled metabolic disease on cerebral vasculopathy, this study provides a level of temporal insight that is not routinely presented. The current results indicate that female OZR maintain normal cerebral vascular reactivity and microvessel density function for a longer period despite the increasingly poor environment. There is also evidence that female OZR may manifest levels of stress and anxiety that are not as commonly present in males, associated with a more rapid development of hypertension within ICA and higher plasma cortisol levels. The combination of the higher perfusion pressure and patterns of cerebral vascular reactivity may result in an increase in the severity of pressure wave penetrance into the deeper cerebral microcirculation, an effect that may be well correlated with both the severity and the timing of an increased emergence of depressive symptoms in the female OZR as compared with age-matched males. What makes these results particularly intriguing is the implication that prolonged maintenance of more normal cerebral vascular endothelial function and reactivity in female OZR with metabolic disease could offer protection against cerebrovascular pathologies such as stroke or transient ischemic attacks. However, it may also predispose females to impaired cognitive behavior emergence at a rate exceeding that in male OZR. Further investigation into these relationships, their underlying mechanisms, and translation to relevant human subjects is warranted.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

The authors gratefully acknowledge the support provided from the National Institutes of Health (R01 DK64668, RR 2865AR), the Canadian Institutes for Health Research (#389769), and the Natural Sciences and Engineering Research Council (Canada; RGPIN-2018-05450). In addition, we also acknowledge the support provided through Center for Cardiovascular and Respiratory Sciences at the West Virginia University Health Sciences Center and via the Research Office from the University of Western Ontario. L.K. is supported by research fellowship from the Natural Sciences and Engineering Research Council.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

F.K. and J.C.F. conceived and designed research; F.K., L.K., I.Z., and J.C.F. performed experiments; F.K., L.K., I.Z., and J.C.F. analyzed data; F.K., L.K., I.Z., S.N.W., D.G., and J.C.F. interpreted results of experiments; F.K., L.K., I.Z., and J.C.F. prepared figures; F.K., L.K., I.Z., S.N.W., D.G., and J.C.F. drafted manuscript; F.K., L.K., I.Z., S.N.W., D.G., and J.C.F. edited and revised manuscript; F.K., L.K., I.Z., S.N.W., D.G., and J.C.F. approved final version of manuscript.