OBESITY INCREASES BLOOD PRESSURE, CEREBRAL VASCULAR REMODELING, AND SEVERITY OF STROKE IN THE ZUCKER RAT

Abstract

Obesity is a risk factor for stroke, but the mechanisms by which obesity increases stroke risk are unknown. Because microvascular architecture contributes to the outcome of stroke, we hypothesized that middle cerebral arteries (MCA) from obese Zucker rats (OZR) undergo inward remodeling and develop increased myogenic tone compared to lean Zucker rats (LZR). We further hypothesized that OZR have an increased infarct following cerebral ischemia and that changes in vascular structure and function correlate with the development of hypertension in OZR. Blood pressure was measured by telemetery in LZR and OZR from 6 to 17 weeks of age. Vessel structure and function were assessed in isolated MCAs. Stroke damage was assessed after ischemia was induced for 60 minutes followed by 24 hours of reperfusion. While mean arterial pressure (MAP) was similar between young rats (6–8 weeks old), MAP was higher in adult (14–17 weeks old) OZR than LZR. MCAs from OZR had a smaller lumen diameter and increased myogenic vasoconstriction compared to those from LZR. Following ischemia, infarction was 58% larger in OZR than LZR. Prior to the development of hypertension, MCA myogenic reactity and lumen diameter as well as infarct size were similar between young LZR and OZR. Our results indicate that the MCAs of OZR undergo structural remodeling and that these rats have greater cerebral injury following cerebral ischemia. These cerebrovascular changes correlate with the development of hypertension and suggest that the increased blood pressure may be the major determinant for stroke risk in obese individuals.

INTRODUCTION

Stroke is the third leading cause of death in the United States and the major source of debilitation among adults.¹ Obesity is a growing epidemic in the United States and is considered a risk factor for stroke.¹ The mechanisms by which obesity affects stroke occurrence or outcome, however, have not been fully elucidated. Hypertension develops in many obese individuals, and according to the Heart Disease and Stroke 2008 Update, hypertension is the greatest risk factor for stroke.¹ While obesity is associated with hypertension and its related end-organ damage, the exact mechanism relating the two cardiovascular risk factors is not completely understood. Considering the coincident occurrence of hypertension and stroke risk in obese patients, it is possible that elevated arterial pressure is a major factor in cerebral vascular injury in the obese population

One potential mechanism by which obesity may increase the risk of stroke is alteration in the perfusion of the cerebral circulation. Cerebral perfusion is regulated through active constrictor and dilator mechanisms and by the physical properties of the cerebral vasculature. Notable determinants of cerebral perfusion that correlate with stroke injury include myogenic tone and vessel structure. Increased myogenic tone has been demonstrated in middle and posterior cerebral arteries from spontaneously hypertensive rats (SHR) as well as vasopressin-deficient rats.^2–4 Interestingly, González et al. demonstrated that six month old but not one month old SHR have increased resting tone in the middle cerebral artery (MCA) compared to their normotensive counterpart the Wistar Kyoto rat.³ Additionally, there is ample evidence of vascular remodeling in the cerebral circulation in several models of hypertension, focusing especially on SHR.^4–8 Of importance, however, is the limitation of these studies to models of severe malignant hypertension. Experimental evidence for cerebral vascular remodeling in more moderate forms of hypertension, such as those with obesity-induced hypertension, is lacking.

A common finding in hypertensive populations is changes in the mechanical and architectural properties of the cerebral vasculature, specifically an inward remodeling of the large arteries such as the MCA.^{6, 9, 10} In addition to increasing the risk of flow obstruction, inward remodeling can be detrimental in ischemic conditions. Following ischemia vessels in the brain are near-maximally vasodilated, and thus, reductions in maximum lumen diameter become a constraint on perfusion. Changes in arterial pressure are well documented to cause deleterious changes in vascular structure; as vessels become stiffer, wall thickness can increase, and lumen diameter can decrease.^{6, 11} As blood flow during ischemia is directly related to maximum lumen diameter, flow will be impaired as lumen diameter is decreased. Whether hypertension is the causal risk factor for alterations in cerebral vascular structure and, subsequently, whether these changes in cerebral vascular structure are the mechanism of increased stroke risk in the obese population is unknown. Therefore, we tested the hypothesis that OZR would have an increased infarct size following cerebral ischemia and demonstrate inward remodeling of the MCA and increased myogenic tone compared to LZR. We further hypothesized that changes in infarct size, vascular structure, and function would correlate with the development of hypertension in OZR.

METHODS

Animals

Six-week old male LZR and OZR were purchased from Harlan and were maintained on a 12 hour light/dark cycle with access to food and water ad libitum. Rats were housed in an American Association for Accreditation of Laboratory Animal Care accredited facility, and all protocols were approved by the Institutional Animal Care and Use Committee. Rats were considered young at an age of 6–7 weeks and adult at 14–16 weeks of age.

Telemetry

Rats were implanted with telemetry transmitters (Data Sciences International, St. Paul, MN) in the abdominal aorta during the week prior to the initiation of the study. Rats were anesthetized using isoflurane in oxygen, and a midline incision was made to expose the abdominal aorta. The aorta was briefly occluded to allow insertion of the transmitter catheter that was secured in place with tissue glue. The transmitter body was sutured to the abdominal wall along the incision line as the incision was closed. The skin was closed with staples that were removed seven days after surgery. Rats were allowed to recover from surgery and returned to individual housing. Individual rat cages were placed on top of the telemetry receivers and arterial pressure wave forms were continuously recorded throughout the study.

Euglycemic Insulin Clamp

Following an overnight fast rats were anesthetized with isoflurane (2%) in oxygen and maintained at 37°C on a heating pad. The carotid artery was catheterized for blood sampling, and the jugular and femoral veins were catheterized for infusion of insulin and glucose, respectively. After measuring baseline blood glucose levels rats were infused with insulin (10 mL/min) via the jugular vein, and blood glucose was measured every 5 minutes for 100 minutes. Although insulin infusion rates are typically adjusted for body weight in this protocol, a constant infusion rate was used in the current study because Schreihofer et al. previously demonstrated that LZR and OZR have comparable plasma volumes.¹² Glucose was infused to maintain a glucose concentration of approximately 120 mg/dL. The average glucose infusion rate over the last 30 minutes to maintain euglycemia is reported and used as an indication of insulin sensitivity.

Middle Cerebral Artery Occlusion

Cerebral ischemia was induced by the intralumenal suture model developed by Longa et al.¹³ Rats were initially anesthetized with isoflurane in an induction chamber, and anesthesia was maintained with 2% isoflurane in oxygen; body temperature was maintained at 37°C. The skull was exposed by a small incision for attachment of a laser Doppler flow probe (Perimed, Stockholm, Sweden) to measure blood flow to the region supplied by the MCA. A midline incision was made to expose the carotid artery. The lingual and thyroid arteries were cauterized, and the external carotid and pterygopalatine arteries were ligated with 6-0 suture. A 3-0 nylon monofilament with a rounded end was inserted into the common carotid artery and was advanced through the internal carotid artery to block blood flow to the MCA where it branches from the Circle of Willis. MCA occlusion was verified by a drop in flow as measured by laser Doppler. After 1 hour of ischemia the filament was pulled back to allow for reperfusion of the MCA. Following 24 hours of reperfusion rats were anesthetized and decapitated, and the brain was removed, sliced into 2 mm sections, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MO) to assess ischemic damage. Brains were fixed in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO), and digital images of brain slices were taken. The percentage of infarction was determined in all brain slices using the following equation:

$$\%\text{Hemisphere}\text{Infarcted}=(({\text{V}}_{\text{L}}-{\text{V}}_{\text{C}})/{\text{V}}_{\text{C}})\ast 100$$

where V_L is the volume of the lesioned hemisphere and V_C is the volume of the control hemisphere.

Middle cerebral artery reactivity and structure assessment

Rats were euthanized with isoflurane and decaptitation, and trunk blood was collected for the measurement of plasma insulin levels (ELISA, Alpco Diagnostics). MCAs were dissected from the brain following decapitation and placed in Krebs buffer (contents in mM: 118 NaCl, 23.8 NaHCO₃, 4.7 KCl, 1.2 MgSO₄, 1.18 KH₂PO₄, 11.1 glucose, 1.9 CaCl₂) on ice. Vessels were mounted on glass micropipettes in a pressure myograph (Living Systems Instrumentation, Burlington, VT) and secured using nylon suture. Only vessels that held pressure at 80 mmHg were used for experimentation. The arteriograph was connected to a pressure servo controller by a pressure transducer, allowing for pressure to be manipulated under zero-flow conditions. A video dimension analyzer connected to a monitor permitted measurement of lumen and outer diameters. Following equilibration at 80 mmHg in Krebs buffer heated to 37°C and gassed with 21% O₂, 5% CO₂, and 74% N₂, the myogenic response was determined by measuring lumen diameter as pressure was increased by 20 mmHg increments over a range of intralumenal pressures (0–140 mmHg). The same measurements were made in the absence of calcium to determine passive vessel structure. Vessels were incubated in calcium-free buffer for at least 1 hour to deplete calcium from the vessels. Complete loss of active tone was verified by confirming the lack of a response in lumen diameter upon addition of papaverine. Comparing the lumen diameters under active and passive conditions, the percentage of myogenic tone was calculated:

$$\%\text{Myogenic}\text{Tone}=(1-({\text{ID}}_{\text{A}}/{\text{ID}}_{\text{P}}))\ast 100$$

where ID_A is inner diameter under active conditions and ID_P is inner diameter under passive conditions.

Statistics

Physiological parameters, glucose infusion rates, and cerebral infarct size were compared between age-matched LZR and OZR using a Students t-test. A two-way ANOVA was used to compare lumen diameter and myogenic tone between LZR and OZR. A p-value less than 0.05 was considered statistically significant. Values are presented as mean ± SEM.

RESULTS

Table 1 provides comparisons of baseline physiological parameters between LZR and OZR. Body weight was dramatically increased in adult OZR compared to LZR. Importantly, there was evidence of increased body weight in OZR as early as 6–7 weeks of age, however, as indicated by %HbA1c, there was no difference in the glycemic load between adult LZR and OZR. The majority of HbA1c measurements in young rats yielded results that were lower than the detection of the kit. Therefore, these values cannot be reported. Plasma insulin levels were increased in both young and adult OZR compared to age-matched LZR, indicating that even at a young age, OZR demonstrate evidence of insulin resistance (IR). Furthermore, there is a difference in insulin sensitivity between LZR and OZR detected by a euglycemic insulin clamp as early as 6–7 weeks old (Figure 1). Although OZR become more severely insulin resistant at an age beyond the scope of the current study, it is important to note that young and adult OZR exhibit a similar degree of IR based on the insulin clamp data. Mean arterial pressure (MAP) was significantly elevated in adult OZR, however, MAP was similar between young LZR and OZR. The time course of the increased blood pressure in OZR is shown in Figure 2. Therefore, young OZR represent a normotensive model of obesity and IR, while adult OZR are obese, insulin resistant, and mildly hypertensive.

Table 1.

Basic Physiological Parameters of Young and Adult LZR and OZR

Parameter	Adult LZR	Adult OZR	Young LZR	Young OZR
Age (weeks)	16.0 ± 0.4	15.8 ± 0.3	7.2 ± 0.2	7.4 ± 0.2
Body Weight (g)	380 ±7	573 ± 17*	202 ± 14	288 ± 15*
Mean Arterial Pressure (mmHg)	95 ± 3	118 ± 5*	95 ± 1	98 ± 1
HbA1c (%)	4.8 ± 0.1	4.8 ± 0.4	<4.0†	<4.0†
Plasma Insulin (μg/L)	0.18 ± 0.05	5.28 ± 0.55*	0.20 ± 0.08	4.39 ± 1.01*
Plasma Cholesterol (mg/dL)	46.1 ± 3.4	78.5 ± 12.4*	26.9 ± 1.7	38.5 ± 3.5*

^*denotes p<0.05 vs. age-matched LZR.

^†HbA1c levels in the young rats were below the limit of detection (4.0%) of the kit used for these studies.

Figure 1.

Average glucose infusion rate required to maintain euglycemia (125 mg/dL) during hyperinsulin clamp in young and adult LZR and OZR. Insulin was infused at a constant rate of 10 μL/min, and blood glucose was measured every 5 minutes for 90 minutes. Values are mean ± SEM of the last 30 minutes of infusion and n ≥ 5 for each group. * denotes p<0.05 vs. age-matched LZR.

Figure 2.

Mean arterial pressure (MAP) in LZR and OZR from 7–17 weeks of age. MAP was measured in conscious rats via telemetry. Values are mean ± SEM of 24 hour averages and n ≥ 6 for each measurement.

The response to cerebral ischemia was compared between adult male OZR and LZR using the middle cerebral artery occlusion (MCAO) technique described by Longa et al.¹³ Rats were exposed to one hour of ischemia, followed by twenty-four hours of reperfusion. OZR had a significantly larger infarct size compared to LZR (10.6 ± 2.0 vs. 27.4 ± 5.5% hemisphere infarcted, LZR vs. OZR, p<0.05), as indicated by the percentage of infarction in Figure 3. Laser Doppler flowmetry was used to confirm that the degree of occlusion was similar between the two groups (58.4 ± 4.6 vs. 57.5 ± 5.2 % change in cerebral blood flow, LZR vs. OZR).

Figure 3.

Response to cerebral ischemia in adult (14–17 weeks of age) LZR and OZR after 1 hour of ischemia and 24 hours of reperfusion. Representative brain slices are shown on top. Values are mean ± SEM and n ≥ 6. * denotes p<0.05 vs. LZR.

Alterations in myogenic tone and MCA structure were assessed to determine whether these important contributors to cerebral blood flow were altered in obesity. The percentage of myogenic tone was calculated from active and passive pressure response curves that were produced using isolated, pressurized vessels of adult male OZR and LZR as described above. Myogenic tone was significantly increased in vessels from OZR from 80–120 mmHg compared to those from LZR (Figure 4A). Additionally, OZR demonstrated a smaller lumen diameter over the entire range of pressures measured compared to LZR (Figure 4B). There was also a decrease in outer diameter in adult OZR and a small but statistically significant decrease in wall thickness (data not shown). These findings suggest that MCAs from OZR undergo an inward remodeling.

Figure 4.

Myogenic tone (A) and lumen diameter (B) in middle cerebral arteries from adult LZR and OZR. Values are mean ± SEM and n ≥ 8. * denotes p<0.05 vs. LZR.

In order to determine whether infarct size, myogenic tone, and MCA structure in OZR are associated with blood pressure, these parameters were assessed in young rats, prior to the increase in MAP. As shown in Figure 5, following one hour of MCAO and twenty-four hours of reperfusion, infarct size was similar between young LZR and OZR (7.5 ± 1.5 vs. 8.3 ± 1.8% hemisphere infarcted, LZR vs. OZR). Additionally, myogenic tone was similar or decreased in MCAs from young OZR, and there was no evidence of inward remodeling of the MCA according to measurements made in isolated vessels (Figure 6). These results indicate that deleterious changes in the cerebral circulation of adult OZR are not present in young OZR prior to the onset of hypertension despite obesity and IR.

Figure 5.

Response to cerebral ischemia in young (6–7 weeks of age) LZR and OZR after 1 hour of ischemia and 24 hours of reperfusion. Representative brain slices are shown on top. Values are mean ± SEM and n ≥ 6.

Figure 6.

Myogenic tone (A) and lumen diameter (B) in middle cerebral arteries from young LZR and OZR. Values are mean ± SEM and n ≥ 8. * denotes p<0.05 vs. LZR.

DISCUSSION

The goal of the current study was to test the hypothesis that obesity and hypertension in the Zucker rat are associated with changes in cerebral vascular function and structure and increased stroke-induced injury. The key findings of this study are that adult OZR with moderate hypertension and severe IR present with increased cerebral vascular myogenic tone, inward cerebral vascular remodeling and increased cerebral tissue death following ischemia/reperfusion injury. Moreover, juvenile OZR, obese and insulin resistant but not yet hypertensive, show none of these deficits. These findings indicate that prolonged, moderate hypertension, as evident in the adult OZR, is a risk factor for cerebral vascular dysfunction and stroke. Relevant to these observations are the caveats of the model and the methods, effects of obesity on the cerebral circulation, and the role of hypertension in stroke.

OZR have been widely employed to investigate the cardiovascular effects of obesity and IR. The novel aspect of the current study, however, is that OZR demonstrate increased susceptibility to cerebral ischemia/reperfusion injury. Other rodent models of obesity have also implicated that obesity worsens the response to ischemia. A recent study by Terao et al. demonstrated that leptin deficient ob/ob mice have a larger infarct size following MCA occlusion with an intralumenal suture than their litter-mate controls.¹⁴ Additionally, photochemically-induced thromobosis in the MCA resulted in increased stroke damage in ob/ob mice and diet-induced obese mice compared to controls.¹⁵ One caveat of the current study is that blood gases were not measured during ischemia in the current study. While differential regulation of blood gases and pH cannot be ruled out as an explanation of differences in stroke outcome, it should be noted that anesthesia during the ischemic period in the current study was brief; rats were conscious and breathing normally during the majority of the ischemic period, as rats were allowed to wake up during ischemia, and during the entire 24 hour reperfusion period.

It is important to consider that although the cause of obesity in the OZR is not common among humans, the phenotype parallels human obesity in many ways. These rats have increased triglycerides, cholesterol, and insulin and eventually develop diabetes. It must be noted, however, that OZR are morbidly obese, and there is evidence that stroke occurrence is directly related to body mass index (BMI). A prospective study by Kurth and colleagues reported that there is a significant increase in the incidence of total stroke, both ischemic and hemorrhagic, with each unit increase in BMI, even after correcting for hypertension and diabetes.¹⁶

The impact of obesity and it’s associated risk factors on the cerebral vasculature is incompletely understood. Several studies have reported that adult OZR have impairments in endothelium-dependent vasodilation of many vascular beds, including the cerebral circulation.^17–20 In addition to reporting endothelial dysfunction of the MCA, Phillips et al. described that OZR display evidence of increased constriction to 5-hydroxytryptamine and an increased myogenic response and increased myogenic tone at baseline pressures.²⁰ These results are consistent with our findings of increased myogenic tone in OZR over a range of intralumenal pressures. Contrary to what is reported here, Stepp et al. described remodeling of the hindlimb but not the cerebral circulation of OZR.²¹ The reason for this discrepancy is unclear, but the lumen diameter measurements from Stepp et al. show a trend towards decreased diameter with a smaller sample size. Another novel aspect of the current study is the assessment of cerebrovascular structure and function and the response to cerebral ischemia in young OZR.

Clinical and statistical evidence indicates that hypertension is the single greatest risk factor for stroke.¹ A relationship between blood pressure and stroke has been clearly defined in experimental models, however, the majority of studies examining the effect of blood pressure on stroke have been in malignantly hypertensive models such as the spontaneously hypertensive rat.^22–24 Less clear is the effect of a more moderate increase in pressure. The temporal correlation between blood pressure and ischemic injury, as well as alterations in cerebrovascular structure and tone, presented in the current study suggest that even a modest increase in pressure over time in the context of obesity may be detrimental to the cerebrovasculature and worsen the response to cerebral ischemia.

One caveat to consider is that hypertension may be necessary for the cerebrovascular complications in obesity but not sufficient in itself to drive the increased vascular and stroke injury. The extent to which other risk factors present in obesity, such as IR, amplify the role of increased arterial pressure in obesity remains to be determined. The results from the euglycemic hyperinsulinemic clamp experiments suggest that the extent of IR in young and adult OZR is similar. Thus, a worsening of IR cannot explain the observed changes in the cerebrovasculature of OZR. Although IR is not worse in adult OZR than young OZR, the importance and contribution of long-term sustained IR in this model is not clear. Moreover, metabolic perturbations such as hyperinsulinemia and changes in plasma lipids may have similar long-term consequences on severity of stroke. The potential interaction of metabolic and hemodynamic factors in cerebral vascular function in obesity is an important area for future study.

PERSPECTIVES

This study further underscores the relationship between obesity and stroke. Furthermore, it provides new evidence that obesity is a risk factor for pathological alterations in the cerebral vasculature. Our results suggest that a chronic, moderate increase in blood pressure may be a major contributor to increased stroke risk and injury in the obese population. Further studies are required to determine the exact role of obesity-induced hypertension in the pathology of stroke, as well as the contribution of other metabolic factors such as chronic IR.