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LOMA LINDA UNIVERSITY School of Medicine in conjunction with the Faculty of Graduate Studies
______
Prenatal Undernutrition, Metyrapone, and the Cerebrovasculature
by
Lara Durrant
______
A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Physiology
______
June 2017
© 2017
Lara Durrant All Rights Reserved Each person whose signature appears below certifies that this dissertation in his/her opinion is adequate, in scope and quality, as a dissertation for the degree Doctor of Philosophy.
, Chairperson William J Pearce, Professor of Physiology
John Buchholz, Professor of Pharmacology
Omid Khorram, Associate Professor of Reproductive Endocrinology
Wolff Kirsch, Professor of Biochemistry
Eugenia Mata-Greenwood, Assistant Professor of Pharmacology
Lubo Zhang, Professor of Pharmacology
iii ACKNOWLEDGEMENTS
I would like to thank Dr. Pearce for his mentorship. You have taught me over the years how to read and write analytically, how to conduct scientific research, and, of course, how to keep a sense of humor throughout the journey. Thank you for never letting me forget about the “philosophy” part of the PhD.
Thank you as well to my committee members for their thoughtful and timely feedback. I am grateful that you have participated in this process with me.
I would like to thank my lab mates from as Risley Hall. To Billy and Dr.
Buchholz, for first welcoming me to Loma Linda as a technician and for encouraging me to pursue graduate studies.
Thank you as well to all of the members of Lab A, both old and new. You all have truly become like a family to me. Special thanks go especially to Naomi Franco for being an excellent lab partner “in crime” as well as to Desirelys Carreon for her excellent technical skills. I will miss you all as I start off on a new adventure.
Lastly, thank you to my friends and family, especially my mom and dad. You have supported me and kept me sane over the past few years. It hasn’t been easy, but you’ve always been there for me. :-)
iv CONTENT
Approval Page ...... iii
Acknowledgements ...... iv
List of Figures ...... viii
List of Abbreviations ...... ix
Abstract ...... xi
Chapter
1. Introduction ...... 1
Fetal Programming and Adult Origins of Adult Disease ...... 1 Nutritional Programming ...... 2 Stress Hormones and the HPA Axis ...... 3 Glucocorticoids and Pregnancy ...... 4 Glucocorticoid Hypothesis of Food Restriction...... 5 Metyrapone ...... 6 Myogenic Tone ...... 7 Vascular Smooth Muscle Cells ...... 8 Neurovascular Regulation and the Cerebrovascular Anatomy ...... 10 Perinatal Hypoxia-Ischemic Encephalopathy ...... 11 Clinical Significance ...... 13 Purpose and Central Hypothesis ...... 14 References ...... 17
2. Maternal Food Restriction Modulates Cerebrovascular Structure and Contractility in Adult Rat Offspring: Effects of Metyrapone ...... 22
Abstract ...... 23 Introduction ...... 23 Materials and Methods ...... 26
General Preparation ...... 26 Measurement of Smooth Muscle Calcium Concentration ...... 28 Experimental Protocols ...... 29 Fluorescent Immunohistochemistry ...... 29 Chemical Reagents ...... 30 Data Analysis and Statistics ...... 30
Results ...... 31
v Effects of Changes in Pressure on Maximum Passive Diameter in Control, MFR and MFR +Metyrapone MCAs ...... 32 Effects of Changes in Pressure on Myogenic Contractility in Control, MFR and MFR +Metyrapone MCAs ...... 34 Effects of Changes in Pressure on Potassium-Induced Contractility in Control, MFR and MFR +Metyrapone MCAs ...... 37 Effects of Maternal Food Restriction and Metyrapone on Contractile Protein Colocalization and Smooth Muscle in MCAs ...... 40
Discussion ...... 43 Acknowledgements ...... 50 References ...... 52
3. The Impact of Prenatal Glucocorticoid Inhibition on Cerebrovascular Structure and Function in a Mild Model of Hypoxia-Ischemia ...... 58
Abstract ...... 59 Introduction ...... 59 Materials and Methods ...... 63
General Preparation ...... 63 Measurement of Smooth Muscle Calcium Concentration ...... 65 Fluorescent Immunohistochemistry ...... 66 ELISA ...... 67 Chemicals and Reagents ...... 67 Data Analysis and Statistics ...... 67
Results ...... 68
Plasma Corticosterone in a Model of Mild Hypoxia Ischemia ...... 69 Effects of Changes in Pressure on Maximum Passive Diameter and Compliance in Sham, Ischemia, Sham+Metyrapone and Ischemia+Metyrapone Pup MCAs ...... 71 Changes in Diameter, Calcium, and Calcium Sensitivity Induced by Pressure and K+ in Pup MCAs ...... 74 Effects of Ischemia ± Metyrapone on Markers of Smooth Muscle Cell Differentiation in Pup MCAs ...... 78 Effects of Ischemia ± Metyrapone on Colocalization of MHC Isoforms with Smooth Muscle α-Actin ...... 80
Discussion ...... 83 Conclusions ...... 89 Acknowledgements ...... 89 References ...... 90
4. Validation of a Mild Ischemia Model ...... 95
vi
Introduction ...... 95 Materials and Methods ...... 95
FluoroJade C ...... 96 C-fos ...... 97 Glial Fibrillary Acidic Protein (GFAP) ...... 97 Ionized Calcium-Binding Adapter (IBA-1) Protein ...... 98 Magnetic Resonance Imaging (MRI) ...... 98
Results & Discussion ...... 99 Conclusions ...... 112 References ...... 113
5. Conclusions and Future Directions ...... 116
vii FIGURES
Figures Page
1. Chemical Structure of Metyrapone ...... 7
2. Proposed Mechanism of MFR and Ischemic Vulnerability ...... 14
3. Effects of Maternal Food Restriction on Passive Arterial Diameter and Compliance ...... 33
4. Effects of MFR and Metyrapone on Myogenic Pressure-Diameter Relations ...... 36
5. Effects of Changes in Pressure on Potassium-Induced Contractility in Control, MFR, and MFR+Metyrapone MCAs ...... 39
6. Effects of Maternal Food Restriction and Metyrapone on Contractile Protein Colocalization and Smooth Muscle in MCAs, Composite ...... 41
7. Effects of Maternal Food Restriction and Metyrapone on Contractile Protein Colocalization and Smooth Muscle in MCAs, Quantitative Analysis ...... 42
8. Plasma Corticosterone Levels in a Model of Mild Ischemia ...... 70
9. Passive Diameter and Incremental Compliance in Ischemia and MET arteries...... 73
10. Changes in Diameter, Calcium, and Calcium Sensitivity Induced by Pressure and K+ in Ischemia and MET arteries ...... 77
11. Effects of Ischemia ± Metyrapone on Markers of Smooth Muscle Cell Differentiation ...... 79
12. Effects of Ischemia ± Metyrapone on Colocalization of MHC Isoforms with Smooth Muscle α-Actin ...... 82
13. Measurements of Cerebral Infarct in a Model of Mild Ischemia ...... 100
14. Behavior Data from Sham vs Ischemia Animals in Mild Hypoxia- Ischemia: Righting Reflex, Geotaxis Reflex, and Open Field Exploration ...... 102
15. 4-Way Histological Analysis with GFAP, IBA-1, c-fos, and FJC of Sham vs Ischemia Pup Brains ...... 104
16. Comparison of T2 and ADC in Sham vs Ischemia...... 107
17. Body Weights of Pups at P11/12 Post-Treatment ...... 110
viii ABBREVIATIONS
11-BHSD2 11β-Hydroxysteroid dehydrogenase isozyme 2
ACTH Adrenocorticotropic Hormone
AIF-1 Allograft inflammatory factory
BBB Blood-brain barrier
CBG cortisol binding globulin
C-FOS Proto-oncogene from the fos family
CRH corticotropin-releasing hormone
CV cresyl violet
EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid
FJ-C FluoroJade-C
GC Glucocorticoid
GFAP Glial Fibrillary Acidic protein
GRE glucocorticoid response element
H&E hematoxylin and eosin staining
HIE hypoxic-ischemic encephalopathy
HPA hypothalamic pituitary axis
IBA1 Ionized calcium-binding adapter molecule 1 (see AIF-1)
ICH Ischemia/ intracerebral hemorrhage
IUGR intrauterine growth restriction
MCA middle cerebral artery
MET Metyrapone
MFR maternal food restriction
MLC myosin light chain
MLCK myosin light chain kinase
ix MLCP myosin light chain phosphatase
MMP matrix metalloproteinases
MRI Magnetic Resonance Imaging
NSPC Neural stem cells
NM-MHC, SMemb non-muscle myosin heavy chain, embyronic myosin heavy chain
P# postnatal day #
PMCA plasma membrane Calcium ATPase
PSS physiological saline solution
RAAS renin-angiotensin system
ROCK Rho-associated protein kinase
SERCA sarcoplasmic reticulum Calcium ATPase
SM1, SM2, SM-MHC smooth muscle myosin heavy chain
SMaA smooth muscle alpha actin
SMC smooth muscle cell
TIMP metallopeptidase inhibitor tPA tissue plasminogen activator
VEGF vascular endothelial growth factor
VSMC vascular smooth muscle cell
x ABSTRACT OF THE DISSERTATION
Prenatal Undernutrition, Metyrapone, and the Cerebrovasculature
by
Lara Durrant
Doctor of Philosophy, Graduate Program in Physiology Loma Linda University, June 2017 Dr. William J. Pearce, Chairperson
The link between intrauterine environmental conditions and adult cardiovascular system is well established. Independent of lifestyle factors such as poor diet and exercise habits, individuals who have been exposed to stressful conditions in utero show an increased risk of health problems such as hypertension, stroke, and type II diabetes. In support of the Fetal Origin of Adult Disease hypothesis, many labs have reported permanent anatomical and physiological changes associated with fetal stress and nutrient deficiency, with a focus on organ systems such as the kidney and heart. One key idea proposed by many of these studies is the glucocorticoid hypothesis, which suggests that fetal stress develops at least in part by fetal overexposure to stress hormones, like glucocorticoids, during gestation. However, few studies have looked at the glucocorticoid hypothesis in the context of blood vessels of the brain. In our first study (cohort 1), pregnant female Sprague-Dawley rats were exposed to 50% maternal undernutrition during the last ten days of gestation. One subgroup was also administered Metyrapone, a corticosterone synthesis inhibitor, during gestation. After delivery, the pups were allowed to grow to maturity at 8-months of age, at which time their middle cerebral arteries were harvested and examined using a combination of vessel bath myography and confocal microscopy. In our second study (cohort 2), pregnant female Sprague-Dawley rats were
xi exposed to Metyrapone (±). On postnatal day 9-10, the rats then underwent Hypoxia-
Ischemia (±) (unilateral carotid ligation combined with 1.5 hours 8% hypoxia after 24 hours). The goal of both studies was to determine whether glucocorticoids would increase ischemic vulnerability, an important marker of vascular health. Our results demonstrated that food restriction as well as perturbations to early glucocorticoid exposure are critical to the development of the cerebrovasculature, and that pharmacological glucocorticoid inhibition during gestation can alter the vulnerability to mild hypoxic-ischemic injury.
Future studies are currently in progress to determine the ultimate effects of food restriction on the cerebrovasculature during the neonatal period.
xii CHAPTER ONE
INTRODUCTION
Fetal Programming and Adult Origins of Adult Disease
The Fetal Origins of Adult Disease hypothesis was first proposed and developed by Dr.
David Barker, an epidemiologist, based on historical data collected from the Dutch Famine of
1944-45 (4). During World War II, the food supply to parts of the western Netherlands was severely limited, triggering the start of what would become the Hunger Winter in September of
1944. Many adults began to receive less than 1000 kcal per day. In May 1945, after many months of hardship and strict food rationing, the food supply lines were restored and the famine ended.
However, its legacy would extend far into the future. By studying the birth records, it was found that infants whose mothers had been exposed to the famine were smaller at birth and showed an increased risk of developing type II diabetes, obesity, stroke and hypertension as adults (2), and that these effects persisted through several generations (14). These findings have since been duplicated in multiple laboratory settings and have been interpreted to support the general theory of “fetal programming”.
The main principle behind fetal programming is that a fetus retains a certain level of phenotypic plasticity as it develops, and is able to actively sense, adapt, and react to in utero stressors such as hypoxia, poor nutrition, or maternal infections (51). Subsequently, the fetus is better able to survive, even under suboptimal environmental conditions. However, since these adaptations often take the form of tissue remodeling via epigenetic mechanisms, they may result in permanent anatomical changes (33), particularly if they occur during a critical developmental window. Further, it is believed that these changes can predispose the fetus to later disease. Hence, what may be adaptive for an infant’s short term survival may not be optimal for their long-term health. In regard to specific mechanisms, glucocorticoid programming is considered to be one of the main effectors that play a role in fetal programming. Environmentally-induced epigenetic
1 changes, such as those induced by food restriction or maternal nicotine use, are also thought to be
key players in fetal programming. Some examples of epigenetic mechanisms include altered promoter methylation, histone acetylation, and miRNA synthesis and activity (9).
Nutritional Programming
A growing fetus needs a steady source of nutrients to support its development. When nutrient flow is interrupted or compromised in any way, the fetus must adapt. Many efforts have been made to explain how these adaptations might occur. A key challenge to the study of maternal food restriction (MFR) is the wide variety of models that are found in the literature (32,
52). There are models that selectively examine the effects of micronutrient (vitamins, trace nutrients, etc.) vs. macronutrient (protein, fat, carbohydrate) deprivation. Protein vs caloric restriction models have also been extensively studied. Generally speaking, programming effects, like food restriction, can be thought of through a “pharmacodynamics” lens such that the characteristics of the insult— dose, degree of exposure, individual characteristics of the developing fetus, etc.- are all important. Despite the variety of different models, however, the resulting phenotype is quite consistent, occurring independently of other lifestyle factors such as a
Western or high fat diet. Typically, the MFR phenotype contributes to an altered pattern of fetal and placental growth (2), resulting in low birth weight offspring that later develop high blood pressure as adults (39). Because of the consistent effects of MFR on blood pressure, considerable research has focused on the kidney and the renin angiotensin aldosterone system (RAAS), and food restriction has been attributed to impaired nephrogenesis and kidney growth (34). Other organ systems, and in particular the brain, have remained understudied. One explanation for this might be the popularity of the brain sparing theory which proposes that a growing infant can compensate for decreased nutrients by increasing blood flow to critical organs such as the brain and heart (3, 11, 15), with the assumption that the brain would be protected. Supporting this
2 assertion are the observations that brain size relative to body size tends to be preserved even in
very small infants. Hemodynamic changes also occur in the vasculature that support brain
sparing, such as altered contractility levels in the carotid artery in response to maternal food
restriction (5). What has been called into question is not that it occurs, but rather what function it serves and whether it truly protects the brain (46, 58). In addition to brain sparing effects, vascular effects mediated by food restriction have also been reported, and MFR-induced changes in vascular structure have been noted in the aorta, the femoral artery, and the mesenteric artery
(21, 29, 53).
Stress Hormones and the HPA Axis
The HPA axis is a major endocrine feedback system responsible for the long-term stress response that helps to coordinate the actions between three organs, the hypothalamus, the pituitary gland, and the adrenal glands. Briefly, the hypothalamus produces corticotrophin releasing hormone (CRH), stimulating the anterior pituitary to produce adrenocorticotropic hormone (ACTH), which in turn stimulates the adrenal cortex to produce glucocorticoid hormones (cortisol in humans, and corticosterone in rats). Rats also secrete small amounts of cortisol (17). Cortisol and corticosterone both have negative feedback effects on CRH and ACTH production, thus limiting their own production. The HPA axis plays an important role in maintaining homeostasis.
Glucocorticoid hormones are essential for life, rise and fall in a circadian manner, and play a key role in cardiovascular function, the immune system, and metabolism (40). Cortisol has wide-reaching effects within the body. Once produced, glucocorticoids (GCs) may enter the circulation where they typically bind to corticosterone-binding globulin (CBG). Because GCs are fat-soluble hormones, they are able to pass through cell membranes and bind to intracellular glucocorticoid receptors (GR). The bound version of GR then interacts with glucocorticoid
3 response elements (GRE) as a ligand bound transcription factor in the cell’s nucleus in order to
regulate gene transcription. Only a small number of GREs are actually bound by the GR at any
one point in time, and the specific GREs that the GR binds to are often tissue-specific and may rely on chromatin availability. This is known as the classical pathway and, like many fat-soluble hormones, exhibits a slower response time as new protein takes time to be translated. There are also non-classical effects of GCs on cells, some of which may occur more quickly. They are thought to proceed through the activity of various kinases such as MAPK and AKT. There are also a number of GR splice variants, further complicating our understanding of the literature.
Glucocorticoids can potentially have an effect on every tissue and the GR can be found in nearly every tissue including vascular smooth muscle cells (VSMCs). Glucocorticoids also bind to mineralocorticoid receptor (MR). Because of their wide reaching effects, there is evidence that glucocorticoids can contribute to vascular changes, though they may not be the only player, and glucocorticoid-independent mechanisms may also be important. Glucocorticoids may potentiate the response to catecholamines leading to an increased response to norepinephrine within
VSMCs, as well as regulate calcium homeostasis (57).
Glucocorticoids and Pregnancy
The placenta is a specialized organ that forms only during pregnancy that begins to
develop upon implantation of the blastocyst into the uterine endometrium. The placenta forms the
natural barrier between mother and baby. Its core purpose is multi-purposed. The placenta prevents maternal and fetal blood from mixing, regulates the exchange of oxygen and nutrition, it allows for temperature homeostasis, and controls waste removal for the fetus. It also performs a number of protective functions including hormone regulation. Glucocorticoids play a critical role during pregnancy. Cells of the placenta are a major source of CRH production during pregnancy, which helps to start the signal cascade for parturition (30). Other functions include the actions of
4 11-ß hyrdroxy steroid dehydrogenase isozyme 2 (BHSD2) and its ability to convert active cortisol
to its inactive form (44). A major obstacle to the study of maternal and fetal endocrinology is that
the fetal HPA axis, the maternal HPA axis, and hormone regulation by the placenta can all
potentially influence each other. This becomes especially important when speaking about specific
pathophysiological conditions during pregnancy, such as fetal stress. A hallmark feature of an
intrauterine growth restricted fetus is the presence of a larger than normal placenta, which can be
viewed as an adaptive response to the low-nutrient conditions with the larger placenta, i.e.
increased surface area, attempting to compensate for the low substrate availability. Conceptually,
the placenta can be viewed as the main filter through which the fetus evaluates its environment,
such that any disease or nutrient-deficient state that interrupts nutrient or hormone signaling of
the placenta could potentially cause a reaction in the fetus.
Glucocorticoid Hypothesis of Food Restriction
The precise mechanisms by which maternal food restriction exerts its deleterious
physiological effects remain unknown. However, glucocorticoid effects via alterations of the
hypothalamic pituitary adrenal (HPA) axis have been proposed as a major contributor (37).
Known as the “glucocorticoid hypothesis,” the idea is that maternal stress leads to fetal stress by
allowing the fetus to become exposed to high levels of maternal glucocorticoids. In a normal
pregnancy, glucocorticoid levels can become quite high. Typically, the fetus is protected from
these high levels by the placenta. The main barrier is the enzyme 11 ß-HSD2, which is thought to
protect the fetus by converting cortisol to its inactive form cortisone (in the rat, corticosterone is
converted to 11-dehydrocorticosterone) (36, 44). However, it has been shown that this
mechanism may become compromised in food restricted offspring, leading to excess
glucocorticoid exposure. Maternal administration of antenatal glucocorticoids produce a similar phenotype compared with MFR models, and also result in adverse health outcomes in the long-
5 term (6). Hypertension is one of the most frequently reported characteristics of glucocorticoid
programming, illustrating the point that the vasculature appears to be especially targeted by these
developmental changes. Despite extensive evidence, however, the glucocorticoid hypothesis is
controversial, and some labs have reported that programming changes can occur in the absence of
elevated glucocorticoid levels (12, 24). Hence, there remains the possibility that glucocorticoids may play an important, but nonessential role, in the programming effects of food restriction.
Metyrapone
Metryapone, or metopirone, (see Figure 1 for its chemical structure) is a
reversible cytochrome P-450 11 ß-HSD hydoxylase inhibitor that acts on the
corticosterone synthesis pathway by inhibiting the synthesis of steroid 11-ß-
monooxygenase. Metyrapone is used clinically to treat Cushing’s syndrome and to
diagnose adrenal insufficiency. Metyrapone has been used as an effective way to dampen
glucocorticoid levels during pregnancy so as to study the long-term programming effects
and mechanisms of excess glucocorticoid (GC) exposure in utero. (38, 42). Several
studies have examined the effects of MET-only treatment during pregnancy. Female rats
whose mothers received Metyrapone were shown to have slightly longer estrous cycles
and delayed puberty when compared to controls (48). However, neither embryonic
weight nor allantoic size differed when Metyrapone was administered in a pig model
from days 14 through 19 (54). Metyrapone, when administered during pregnancy is well
tolerated (19) and results in otherwise healthy offspring with minor differences which
could be expected from its effects on glucocorticoid synthesis. With only minimal side
effects, Metyrapone has been shown to be highly effective at lowering plasma
glucocorticoid levels during pregnancy (26). This combination of properties have
6 contributed to its popularity as an important tool in understanding the role of GCs during
pregnancy.
Figure 1. Chemical Structure of Metyrapone.
Myogenic Tone
Although the cardiovascular system has been proposed as a major target of food
restriction-related programming, the cerebrovasculature has received little attention in the
literature. Brain tissue is very metabolically active and cannot survive long without a steady
blood flow of nutrients. Effective maintenance of perfusion by blood vessels is critical to brain
function. so it is not surprising that cerebral blood vessels have developed a number of features
that allow them fulfill this purpose. Myogenic tone is one such mechanism that appears to be
particularly important in cerebral arteries. Myogenic tone (also known as the Bayliss effect) is an
intrinsic property of blood vessels, usually arteries or arterioles, that helps to ensure that tissue perfusion remains constant despite pressure changes (13). One of the main functions of myogenic tone, thus, is to regulate blood flow, thereby preventing ischemia due to low blood pressure
(hypoperfusion) or tissue damage due to high blood pressure (hyperperfusion) (47). Myogenic tone is especially important in the vasculature of the brain. While vascular resistance may be modified by sympathetic and endothelial effects, the myogenic response is independently
7 controlled by vascular smooth muscle cells. Myogenic tone can be thought of as an indicator of
overall vascular health which may be altered during various pathological conditions, including
stroke and hypertension (10). Because its functionality can be compromised by certain stressful
conditions, understanding its underlying mechanisms is critical.
The vasculature of young animals is functionally and structurally different than that of
the adult, and certain age-related changes need to be taken into account. First, the dynamic range of immature or developing blood vessels is much smaller. Outside of that range, blood flow is only passively (not actively) controlled by myogenic mechanisms (8). There is also evidence that
a smaller number of contractile cells in neonatal arteries are doing proportionally more of the
work compared to adult arteries (7). Given these findings, the myogenic responses during these
key developmental periods may be more sensitive to injury. It isn’t difficult to imagine how an
infant whose brain is subjected to sudden spikes or drops in blood pressure due to an impaired myogenic response may be more vulnerable to ischemic injury. This innate vulnerability may be one reason as to why stroke rates are higher during the perinatal period, occurring at about 1 in
2300 to 1 in 5000 live births (43). By comparison, adults, especially among elderly populations, have a higher rate of stroke. Even so, the potential long-term impact of neonatal stroke should not be underestimated. Hence, it is critical to understand these age-related differences and how they may specifically effect the developing brain.
Vascular Smooth Muscle Cells
Vascular smooth muscle cells (VSMCs) are critical to understanding the myogenic
response (13). Simply put, VSMCs provide the key structures by which a contraction can occur.
Smooth muscle cell contraction proceeds largely through Ca++ dependent mechanisms and has been extensively studied (49). SMC contraction consists of three main phases: 1) activation of receptors by hormones or neurotransmitters, which activate a number of GTP binding proteins, 2)
8 changes in Ca++ levels either by influx from the extracellular space, release from the sarcoplasmic
reticulum, sequestration within the sarcoplasmic reticulum, or extrusion of calcium into the
extracellular space, and 3) changes in myosin light chain phosphorylation (25). Typically, it is
accepted that phosphorylation of myosin light chain (MLC) by myosin light chain kinase
(MLCK) or de-phosphorylation by myosin light chain phosphatase (MLCP) can selectively control the level of contraction or dilation in SMCs which provides calcium sensitization. A number of other factors can further modulate and fine-tune the overall Ca++ sensitivity of a vessel,
++ ++ including Rho Kinase (ROCK) (50). The amount of intracellular calcium ([Ca ]i) and the Ca
++ ++ sensitivity together control force generation, and the levels of [Ca ]i and Ca can be selectively modified to meet the requirements of the vessel. Another key point is that some types of smooth muscle cells, particularly immature SMCs, do not participate in contraction and force generation.
Indeed, it has been found that SMCs can exist in a variety of non-contractile phenotypes including proliferative and synthetic stages. There is a dynamic equilibrium between the many types of VSMCs and VSMCs retain the ability to differentiate or dedifferentiate based on changing conditions (41). They also have the ability to change their local environment through the production of extracellular matrix components; certain VSMCs may have more fibroblast-like properties. While some cells from each stage will likely be found in any given tissue, a number of factors such as age and environment appear to play a role in the overall distribution of different cell types. For example, it has been reported that SMCs may change phenotypes in response to tissue damage or injury (49). This may potentially explain the findings that both maternal food restriction and glucocorticoids appear to influence extracellular matrix remodeling (27, 28), perhaps by altering the phenotype of VSMCs. Thus, a change in SMC phenotype, no matter the initial cause, could in turn alter the myogenic response and, subsequently, may affect an artery’s ability to auto-regulate.
9 Neurovascular Regulation and the Cerebrovascular Anatomy
As alluded to previously, the brain is a highly metabolically active tissue that is extremely energy sensitive. Even a short period without oxygen, as little as five minutes, can lead to neuronal death. Fortunately, the vascular anatomy provides an excellent source of redundancy in the form of the Circle of Willis. Even if one side is occluded, the collateral perfusion can often compensate. However, there are some regions of the brain such as the cerebral cortex, hippocampus, and sub-ventricular zone that may be more sensitive than others, perhaps due to less collateral backup flow. One important concept is that of functional hyperemia and the idea of neurovascular coupling. At the most basic, it is the careful matching of neural activity with the proper degree of vascular perfusion. Examples include when a person exercises or when they are asleep. Highly metabolically active areas receive more perfusion in this model. This is accomplished partly by way of cerebral autoregulation. Again, while the cerebral vasculature can be partly modulated by signals from neurons, the endothelium, and various other hormones, a main contributor is myogenic tone. In the brain, there exists a normal auto regulatory range where the flow can be dynamically controlled. Problems begin to occur when that auto regulatory range is shifted too far to the right or left. High blood pressure is one such condition under which the range of flow is no longer properly regulated. Hence, if blood pressure fluctuates too much, the protective mechanisms no longer work, exposing the tissues to hypo- or hyperperfusion. These mechanisms can also become damaged during and after stroke, such as with vessel vasospasm.
Factors such as hypertension, diabetes, and acute ischemia may all act as vascular risk factors, altering the structure and function of a vascular bed and impairing the endogenous protective mechanisms, thereby worsening the outcome should an ischemic event occur. A healthy vascular bed, on the other hand, may provide some degree of resistance to disease or injury.
10 Perinatal Hypoxic-ischemic Encephalopathy
Perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity that results in lifelong disability (1, 31), with neonatal stroke averaging about $50,000 per case in the first five years of life for medical care alone (16). Neonates who suffer perinatal stroke can have long term developmental consequences such as delayed puberty, missed developmental milestones, and intellectual disabilities. Causes of HIE include asphyxia related to birth trauma, maternal infections, and blood clotting disorders including sickle cell anemia have been implicated. Even worse, milder neonatal stroke is often difficult to diagnose with babies typically presenting with generalized symptoms such as lethargy, which could be attributed to a number of conditions.
In an adult stroke patient with evidence of a cerebral thrombosis, or blood clot in the brain, tissue plasminogen activator (tPA) is the treatment of choice if a diagnosis can be made within 4 hours post-stroke (after which point tPA administration is contraindicated due to the risk of cerebral hemorrhage). Endardectomy, or the surgical removal of occluding material from an artery, is also an option if the presence of a lesion can be confirmed via imaging and is readily accessible. However, surgical intervention is highly controversial in infants and children, and would only apply to cases where there is a confirmed blood clot, which may not always be the case. In recent years, there has been evidence that therapeutic hypothermia may be beneficial for perinatal stroke (20). However, as with tPA, there is a narrow therapeutic window, limiting its potential usefulness, and the treatment is dependent on early detection (55). Instead, most treatments for neonatal stroke center around supportive care and the careful monitoring of vitals such as blood pressure and electrolyte balance. More treatment options and detection methods need to be developed.
Research in a laboratory setting has centered around the use of experimental animal models. The most commonly used method for studying neonatal stroke is the Rice Vannucci model in which a rat pup, typically at age P7, undergoes surgery to have one of their carotid
11 arteries ligated (45). They are then allowed to recover for an hour or two before then being
exposed to 8% oxygen. The length of oxygen deprivation typically spans about 1-2 hours and is
the time at which the damage occurs. It is the result of both ischemia (the lack of blood flow)
combined with hypoxia (low oxygen). The Rice-Vannucci model is associated with a relatively
low mortality rate and is characterized by white matter damage, typically in areas of high
myelinogenesis. Animal models, in combination with clinical models, have provided important
information about what happens during an ischemic event. One well-supported theory of HIE injury is the glutamate toxicity theory in which dying neurons release toxic levels of certain neurotransmitters (i.e. glutamate) and Ca++, resulting in more neuronal death (18). While much is known about the signal cascade following an ischemic insult, the targeting of specific neuronal death pathways has not led to any significant advances in therapy options.
One potential therapeutic approach for hypoxia-ischemia that remains relatively unexplored is to target aspects of the cerebrovasculature (such as calcium sensitivity) or the risk factors that lead to impaired vessel health. It has been reported that vascular autoregulation is impaired in premature infants, especially in those infants that are already at risk for ischemia (22,
56). The presence of immature vasculature that is unable to properly auto-regulate may increase the likelihood of an ischemic insult. Targeting specific mechanisms of the vasculature might be helpful after a stroke has already occurred. Another strategy might be to pursue preventative care in the case of high-risk pregnancies. For example, it may be possible to leverage pre-ischemic conditioning mechanisms in the hopes of promoting brain ischemic tolerance. Brain ischemic tolerance is the activation of endogenous neuroprotective mechanisms in response to an otherwise noxious stimulus. Stated simply, ischemic tolerance is a therapeutic exposure to a mild stress may leave a target tissue resistant to a later, more severe, stress (35). In the heart, this has been shown to take place, whereby ischemic resistance is conferred during a critical window of time, protecting the tissue from further damage. Unfortunately, the effect typically wears off within a specific interval of time. With respect to the brain, it may be possible for a chronic low-grade
12 insult such as food restriction or mild hypoxia to induce a more permanent programming effect, thereby conferring some lasting amount of ischemic tolerance to an acute stressor. In either case, endogenous protective mechanisms would likely play a major role in determining the ultimate vulnerability of an individual to an ischemic stroke. The ideal scenario, of course, would be to develop a solution, pharmacological or otherwise, that might influence these pathways without compromising the health of the mother or fetus.
Clinical Significance
This dissertation sought to answer a number of questions including 1) how does food restriction alter early cerebrovascular development, 2) how does nutritional programming alter an infant’s ischemic vulnerability and cerebral autoregulation, and 3) how and in what ways do glucocorticoids mediate these processes. With food insecurity on the rise in certain population sectors (23), it is critical to determine the role that nutritional programming may play in long term health outcomes. With the number of premature infants also on the rise due to advances in neonatal care, it is likely that some of these infants will display signs of intrauterine growth restriction (IUGR) and may be administered glucocorticoids in order to promote fetal lung surfactant production in preparation for birth which may in itself contribute to programming (6).
As these infants are likely to be prime candidates for later adult health problems, our understanding of early life programming must be expanded. A better understanding of the relative contribution of each component (nutrition, age, severity of stroke, etc.) will be key in the search for effective treatment options.
From a basic science perspective, the research for this dissertation was highly innovative and offered an exciting opportunity to study contractility and smooth muscle cell phenotype changes in postnatal cerebral arteries in response to ischemic stroke and maternal undernutrition.
Very little is currently known about the cerebrovasculature of young MFR animals and, to our
13 knowledge, this work, once completed, will be the first to investigate these food restricted pup
cerebral arteries in such detail. Even compared with adult arteries, pup arteries are extremely
small, fragile, and difficult to handle. However, with our carefully developed dissection
techniques and expertise in vessel-bath myography, we have been in a unique position to study both the contractility and functional behavior of these arteries. By using advanced imaging techniques, we have gained insights into the state of the vasculature structure and gained a new
perspective into the differentiation states of the vascular SMCs. Importantly, this research has
and will continue to build a strong foundation for future MFR studies. Together, this work will
contribute greatly to our knowledge of how food restriction may permanently alter the developing
cerebrovasculature.
Purpose and Central Hypothesis
The following studies were based on the core hypothesis that fetal programming,
when induced by exposure to maternal food restriction, causes altered glucocorticoid
responses, that lead to permanent changes in cerebrovasculature structure and function
(see Figure 2). A corollary hypothesis was that endogenous protective mechanisms of the
cerebrovasculature have been compromised due to altered glucocorticoid handling,
leading to an increased susceptibility to ischemic stroke.
Figure 2. Proposed Mechanism of MFR and Ischemic Vulnerability
14 Chapter Two, entitled “Maternal food restriction modulates cerebrovascular structure and contractility in adult rat offspring: effects of Metyrapone”, focused primarily on the long-term effects of food restriction on the cerebrovasculature in adults.
In that study, we found that structure-function relationships of the middle cerebral arteries were permanently altered by 10 days of exposure to a 50% caloric restriction during the final 10 days of gestation. It was also found that certain components related to vascular structure, such as compliance, were influenced glucocorticoid signaling while other components such as K+-induced contractility were largely unaffected.
Chapter Three, entitled “The impact of prenatal glucocorticoid inhibition on cerebrovascular structure and function in a model of mild hypoxia-ischemia”, was a collection of control experiments designed to determine whether the changes to the cerebrovasculature seen at 10-months of age due to food restriction and Metyrapone- dosing (as discussed in Chapter Two) were also seen at in postnatal life. A secondary purpose was to determine what component of these changes were glucocorticoid- dependent and which were glucocorticoid-independent, as well as the degree to which factors should as obesity may have played a role in vascular changes. These experiments did not involve food restriction and instead served to determine an appropriate baseline.
These studies focused primarily on development with the added intent of determining the functional consequences of gestational glucocorticoid withdrawal on the postnatal vascular responses and responses to ischemic injury. Together, these experiments have paved the way for research that will be expanded on in Chapter Four.
Chapter Four, entitled “Validation of a Model of Mild Ischemia”, primarily focused on experimental data that was currently in progress. A main aim of the work in
15 Chapter Four was to validate and develop a new milder and more clinically relevant ischemic injury model, complete with advanced imaging techniques including MRI. In this chapter, experimental data focused on the key features of the ischemic injury through histological analysis, MRI data, and behavior. The discussion also provided some speculation as to the question of how gestational Metyrapone might influence our
Ischemia model.
Chapter Five included some closing remarks and the conclusion. This chapter sought to provide a perspective and framework future experiments. Altogether, this dissertation should provide a solid stepping point towards a survey of the current status of the effects of food restriction on the cererbrovasculature during early and adult life.
16 References
1. Baburamani AA, Ek CJ, Walker DW, and Castillo-Melendez M. Vulnerability of the developing brain to hypoxic-ischemic damage: contribution of the cerebral vasculature to injury and repair? Frontiers in physiology 3: 424, 2012.
2. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, and Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 341: 938-941, 1993.
3. Barker DJ, and Hanson MA. Altered regional blood flow in the fetus: the origins of cardiovascular disease? Acta Paediatr 93: 1559-1560, 2004.
4. Barker DJP. THE FETAL AND INFANT ORIGINS OF DISEASE. European journal of clinical investigation 25: 457-463, 1995.
5. Campbell ME, Williams SJ, Veerareddy S, and Davidge ST. Maternal nutrient restriction reduces carotid artery constriction without increasing nitric oxide synthesis in the late gestation rat fetus. Pediatr Res 58: 840-844, 2005.
6. Chang YP. Evidence for adverse effect of perinatal glucocorticoid use on the developing brain. Korean journal of pediatrics 57: 101-109, 2014.
7. Charles SM, Zhang L, Cipolla MJ, Buchholz JN, and Pearce WJ. Roles of cytosolic Ca2+ concentration and myofilament Ca2+ sensitization in age-dependent cerebrovascular myogenic tone. Am J Physiol Heart Circ Physiol 299: H1034-1044, 2010.
8. Charles SM, Zhang L, Longo LD, Buchholz JN, and Pearce WJ. Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries. Am J Physiol Regul Integr Comp Physiol 293: R737- 744, 2007.
9. Chen M, and Zhang L. Epigenetic mechanisms in developmental programming of adult disease. Drug discovery today 16: 1007-1018, 2011.
10. Cipolla MJ, McCall AL, Lessov N, and Porter JM. Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats. Stroke; a journal of cerebral circulation 28: 176-180, 1997.
11. Cohen E, Baerts W, and van Bel F. Brain-Sparing in Intrauterine Growth Restriction: Considerations for the Neonatologist. Neonatology 108: 269-276, 2015.
12. Cottrell EC, Holmes MC, Livingstone DE, Kenyon CJ, and Seckl JR. Reconciling the nutritional and glucocorticoid hypotheses of fetal programming. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 26: 1866-1874, 2012.
17 13. Davis MJ, and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiological reviews 79: 387-423, 1999.
14. de Rooij SR, Painter RC, Phillips DIW, Osmond C, Michels RPJ, Bossuyt PMM, Bleker OP, and Roseboom TJ. Hypothalamic-pituitary-adrenal axis activity in adults who were prenatally exposed to the Dutch famine. European Journal of Endocrinology 155: 153-160, 2006.
15. Desai M, Crowther NJ, Lucas A, and Hales CN. Organ-selective growth in the offspring of protein-restricted mothers. Br J Nutr 76: 591-603, 1996.
16. Gardner MA, Hills NK, Sidney S, Johnston SC, and Fullerton HJ. The 5-year direct medical cost of neonatal and childhood stroke in a population-based cohort. Neurology 74: 372-378, 2010.
17. Gong S, Miao YL, Jiao GZ, Sun MJ, Li H, Lin J, Luo MJ, and Tan JH. Dynamics and correlation of serum cortisol and corticosterone under different physiological or stressful conditions in mice. PloS one 10: e0117503, 2015.
18. Grow J, and Barks JD. Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts. Clinics in perinatology 29: 585-602, v, 2002.
19. Habib S, Gattineni J, Twombley K, and Baum M. Evidence that prenatal programming of hypertension by dietary protein deprivation is mediated by fetal glucocorticoid exposure. American journal of hypertension 24: 96-101, 2011.
20. Harbert MJ, Tam EW, Glass HC, Bonifacio SL, Haeusslein LA, Barkovich AJ, Jeremy RJ, Rogers EE, Glidden DV, and Ferriero DM. Hypothermia is correlated with seizure absence in perinatal stroke. Journal of child neurology 26: 1126-1130, 2011.
21. Holemans K, Gerber R, Meurrens K, De Clerck F, Poston L, and Van Assche FA. Maternal food restriction in the second half of pregnancy affects vascular function but not blood pressure of rat female offspring. Br J Nutr 81: 73-79, 1999.
22. Howlett JA, Northington FJ, Gilmore MM, Tekes A, Huisman TA, Parkinson C, Chung SE, Jennings JM, Jamrogowicz JJ, Larson AC, Lehmann CU, Jackson E, Brady KM, Koehler RC, and Lee JK. Cerebrovascular autoregulation and neurologic injury in neonatal hypoxic-ischemic encephalopathy. Pediatr Res 74: 525-535, 2013.
23. Ivers LC, and Cullen KA. Food insecurity: special considerations for women. Am J Clin Nutr 94: 1740S-1744S, 2011.
24. Jaquiery AL, Oliver MH, Bloomfield FH, Connor KL, Challis JR, and Harding JE. Fetal exposure to excess glucocorticoid is unlikely to explain the effects of periconceptional undernutrition in sheep. J Physiol 572: 109-118, 2006.
18 25. Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano K, Harada K, Miyamoto S, Nakazawa H, Won KJ, and Sato K. Calcium movements, distribution, and functions in smooth muscle. Pharmacological reviews 49: 157-230, 1997.
26. Khorram O, Ghazi R, Chuang TD, Han G, Naghi J, Ni Y, and Pearce WJ. Excess maternal glucocorticoids in response to in utero undernutrition inhibit offspring angiogenesis. Reprod Sci 21: 601-611, 2014.
27. Khorram O, Han G, Bagherpour R, Magee TR, Desai M, Ross MG, Chaudhri AA, Toloubeydokhti T, and Pearce WJ. The Effect of Maternal Undernutrition On Vascular Expression of Micro and Messenger RNA in Newborn and Aging Offspring. Am J Physiol Regul Integr Comp Physiol 298: R1366-1374, 2010.
28. Khorram O, Khorram N, Momeni M, Han G, Halem J, Desai M, and Ross MG. Maternal undernutrition inhibits angiogenesis in the offspring: a potential mechanism of programmed hypertension. Am J Physiol Regul Integr Comp Physiol 293: R745-753, 2007.
29. Khorram O, Momeni M, Ferrini M, Desai M, and Ross MG. In utero undernutrition in rats induces increased vascular smooth muscle content in the offspring. Am J Obstet Gynecol 196: 486 e481-488, 2007.
30. Kota SK, Gayatri K, Jammula S, Kota SK, Krishna SVS, Meher LK, and Modi KD. Endocrinology of parturition. Indian Journal of Endocrinology and Metabolism 17: 50-59, 2013.
31. Lai MC, and Yang SN. Perinatal hypoxic-ischemic encephalopathy. Journal of biomedicine & biotechnology 2011: 609813, 2011.
32. Langley-Evans SC. Developmental programming of health and disease. Proceedings of the Nutrition Society 65: 97-105, 2006.
33. Langley-Evans SC. Nutritional programming of disease: unravelling the mechanism. Journal of anatomy 215: 36-51, 2009.
34. Langley-Evans SC, Welham SJ, and Jackson AA. Fetal exposure to a maternal low protein diet impairs nephrogenesis and promotes hypertension in the rat. Life Sci 64: 965-974, 1999.
35. Lehotsky J, Burda J, Danielisova V, Gottlieb M, Kaplan P, and Saniova B. Ischemic tolerance: the mechanisms of neuroprotective strategy. Anatomical record (Hoboken, NJ : 2007) 292: 2002-2012, 2009.
36. Lesage J, Blondeau B, Grino M, Breant B, and Dupouy JP. Maternal undernutrition during late gestation induces fetal overexposure to glucocorticoids and intrauterine growth retardation, and disturbs the hypothalamo-pituitary adrenal axis in the newborn rat. Endocrinology 142: 1692-1702, 2001.
19 37. Lingas R, Dean F, and Matthews SG. Maternal nutrient restriction (48 h) modifies brain corticosteroid receptor expression and endocrine function in the fetal guinea pig. Brain Res 846: 236-242, 1999.
38. McMullen S, and Langley-Evans SC. Maternal low-protein diet in rat pregnancy programs blood pressure through sex-specific mechanisms. Am J Physiol Regul Integr Comp Physiol 288: R85-90, 2005.
39. Nuyt AM, and Alexander BT. Developmental programming and hypertension. Current opinion in nephrology and hypertension 18: 144-152, 2009.
40. Oakley RH, and Cidlowski JA. The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease. The Journal of allergy and clinical immunology 132: 1033-1044, 2013.
41. Owens GK, Kumar MS, and Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiological reviews 84: 767-801, 2004.
42. Paek DS, Sakurai R, Saraswat A, Li Y, Khorram O, Torday JS, and Rehan VK. Metyrapone alleviates deleterious effects of maternal food restriction on lung development and growth of rat offspring. Reprod Sci 22: 207-222, 2015.
43. Raju TN, Nelson KB, Ferriero D, Lynch JK, and Participants N-NPSW. Ischemic perinatal stroke: summary of a workshop sponsored by the National Institute of Child Health and Human Development and the National Institute of Neurological Disorders and Stroke. Pediatrics 120: 609-616, 2007.
44. Reynolds RM. Glucocorticoid excess and the developmental origins of disease: two decades of testing the hypothesis--2012 Curt Richter Award Winner. Psychoneuroendocrinology 38: 1-11, 2013.
45. Rice JE, 3rd, Vannucci RC, and Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Annals of neurology 9: 131-141, 1981.
46. Roza SJ, Steegers EA, Verburg BO, Jaddoe VW, Moll HA, Hofman A, Verhulst FC, and Tiemeier H. What is spared by fetal brain-sparing? Fetal circulatory redistribution and behavioral problems in the general population. American journal of epidemiology 168: 1145-1152, 2008.
47. Schubert R, and Mulvany MJ. The myogenic response: established facts and attractive hypotheses. Clin Sci (Lond) 96: 313-326, 1999.
48. Smith JT, and Waddell BJ. Increased fetal glucocorticoid exposure delays puberty onset in postnatal life. Endocrinology 141: 2422-2428, 2000.
49. Somlyo AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994.
20 50. Somlyo AP, and Somlyo AV. Signal transduction by G-proteins, rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522 Pt 2: 177-185, 2000.
51. Swanson JM, Entringer S, Buss C, and Wadhwa PD. Developmental origins of health and disease: environmental exposures. Seminars in reproductive medicine 27: 391-402, 2009.
52. Tarry-Adkins JL, and Ozanne SE. Mechanisms of early life programming: current knowledge and future directions. Am J Clin Nutr 94: 1765S-1771S, 2011.
53. Torrens C, Snelling TH, Chau R, Shanmuganathan M, Cleal JK, Poore KR, Noakes DE, Poston L, Hanson MA, and Green LR. Effects of pre- and periconceptional undernutrition on arterial function in adult female sheep are vascular bed dependent. Exp Physiol 94: 1024-1033, 2009.
54. Trainer PJ. Corticosteroids and pregnancy. Seminars in reproductive medicine 20: 375-380, 2002.
55. van der Aa NE, Benders MJ, Groenendaal F, and de Vries LS. Neonatal stroke: a review of the current evidence on epidemiology, pathogenesis, diagnostics and therapeutic options. Acta Paediatr 103: 356-364, 2014.
56. Vutskits L. Cerebral blood flow in the neonate. Paediatric anaesthesia 24: 22-29, 2014.
57. Yang S, and Zhang L. Glucocorticoids and vascular reactivity. Current vascular pharmacology 2: 1-12, 2004.
58. Zhang YG, Li N, Yang JJ, Zhang T, and Yang Z. Effects of maternal food restriction on physical growth and neurobehavior in newborn Wistar rats. Brain Research Bulletin 83: 1-8, 2010.
21 CHAPTER TWO
MATERNAL FOOD RESTRICTION MODULATES CEREBROVASCULAR
STRUCTURE AND CONTRACTILITY IN ADULT RAT OFFSPRING: EFFECTS
OF METYRAPONE
By
Lara Durrant, Omid Khorram, John N. Buchholz, William J. Pearce
This chapter has been published by American Journal of Physiology- Regulatory,
Integrative, and Comparative Physiology 306: R401-410, 2014.
22 Abstract
Although the effects of prenatal under-nutrition on adult cardiovascular health have been well studied, its effects on the cerebrovascular structure and function remain unknown. We used a pair-fed rat model of 50% caloric restriction from day 11 of gestation to term, with ad libitum feeding after birth. We validated that maternal food restriction (MFR) stress is mediated by glucocorticoids by administering Metyrapone
(MET), a corticosterone synthesis inhibitor, to MFR mothers at day 11 of gestation. At age 8-months, Control, MFR, and MFR+MET offspring were sacrificed and middle cerebral artery (MCA) segments were studied using vessel-bath myography and confocal
microscopy. Colocalization of smooth muscle α-actin (SMαA) with non-muscle (NM)
and SM2 myosin heavy chain (MHC) was used to assess smooth muscle phenotype. Our
results indicate that artery stiffness was increased, pressure-evoked myogenic reactivity
was depressed, and myofilament Ca++ sensitivity was decreased in MFR compared to
control offspring. MCA from MFR offspring exhibited a significantly greater
SMαA/NM colocalization, suggesting that the SMCs had been altered toward a non-
contractile phenotype. MET significantly reversed the effects of MFR on stiffness but
not myogenic reactivity, lowered SMαA/NM colocalization, and increased SMαA/SM2
colocalization. Together, our data suggest that MFR alters cerebrovascular contractility
via both glucocorticoid-dependent and glucocorticoid-independent mechanisms.
Introduction
Based on his studies of the Dutch Famine in 1944, David Barker first proposed
that in utero nutritional stress enhances the risk for adult-onset cardiovascular disease (7).
23 Equally important, the adult offspring of food-restricted mothers also exhibit increased risk for Type II diabetes, hypertension, and obesity (47). Given the increasing incidence of these diseases in many industrialized societies (51), it is possible that at least some of these cases are secondary to maternal undernutrition. In support of this possibility is the rising incidence of food insecurity in many population sectors, including some in industrialized societies (31). Given the relevance of these mechanisms, there have been many recent studies of “fetal origins of adult disease”.
In the search for mechanisms mediating fetal programming secondary to maternal food restriction, an early hypothesis was that these effects were mediated by increases in maternal glucocorticoids (40, 43). Additional evidence further suggested that maternal food restriction modified expression of glucocorticoid receptors in cerebral tissues of their offspring (4, 44). Despite strong evidence for involvement of glucocorticoids in fetal responses to maternal food restriction, however, other studies have suggested parallel involvement of glucocorticoid-independent mechanisms (14), including changes in endothelial function (59), VEGF function (36), angiotensin II levels (50), vitamin D availability (2, 52), oxidative stress (62), and vascular miRNA (35).This diversity of evidence strongly suggests that fetal responses to maternal food restriction involve multiple interacting mechanisms that combine to produce the vascular phenotype typical of offspring from food-restricted mothers.
Interpretation of the literature relevant to maternal food restriction is complicated by the broad variety of models and tissues used in these studies, many of which exhibit markedly different responses. In the kidney, maternal food restriction leads to reductions in kidney size, decreased nephron number, abnormalities in the renin-angiotensin system,
24 and decreased angiogenesis (36, 40, 69). Maternal food restriction also results in smaller
heart sizes at birth, cardiomegaly in adult offspring, and coronary artery remodeling with
fibrosis (9, 33). In the aorta, maternal food restriction can alter aortic structure leading to
increased wall thickness and stiffness (36, 38). In contrast, maternal food restriction
generally “spares” the fetal brain from major effects in the short term (17), but in the
long-term can significantly alter brain structure (18) and function, leading to significant behavioral abnormalities (57). Aside from these general changes in the brain, the specific effects of maternal food restriction on cerebrovascular development and structure remain largely unstudied.
To facilitate studies of the effects of maternal food restriction on the developing fetal cerebral circulation, we have developed a rat model involving 50% caloric restriction during the last 10 days of gestation with normal postnatal feeding(16). This model produces a milder nutritional insult than that obtained with food restriction throughout gestation and early postnatal life, thus increasing the fraction of pups surviving through adulthood (29). Because this model reduces only total caloric intake, it
is also less severe than protein restriction models (5), but still produces the adult-onset
hypertension and obesity characteristic of maternal undernutrition (37). With this model
we have found that maternal food restriction can reduce VEGF expression and
angiogenesis, can increase matrix metalloproteinase expression, and can alter multiple
microRNA species that influence vascular development and function (35-37).
In light of published evidence that Maternal Food Restriction (MFR) alters systemic vascular characteristics in adult offspring (7), the present study explores the hypothesis that MFR also influences vascular structure and function in the cerebral circulation of
25 adult offspring. In particular, the experimental approach examined the functional
consequences of MFR for vascular stiffness and compliance in middle cerebral arteries from 8-month old rats whose dams underwent 50% caloric restriction during the final 10 days of gestation. Separate experiments also examined changes in cerebral artery myogenic reactivity and smooth muscle phenotype attributable to MFR. To test the corollary hypothesis that the vascular effects of MFR arise from altered glucocorticoid signaling, we compared the effects of MFR in offspring from dams treated with or without Metyrapone, an inhibitor of 11-β hydroxylase that lowers circulating levels of
corticosterone. Together, these experiments offered a unique perspective of the adult-
onset cerebrovascular consequences of MFR.
Materials and Methods
General Preparation
All experimental procedures were approved by the Institutional Animal Use and
Care Committees at both the Harbor-UCLA Medical Center and Loma Linda University.
First- time-pregnant Sprague-Dawley rats (Charles River Laboratories, Inc., Hollister,
CA) were housed at constant temperature and humidity with a 12h:12h light-dark cycle.
At day 10 of gestation, rats were provided either an ad libitum diet of standard laboratory chow (Lab Diet 5001, Brentwood, MO: protein 23%, fat 4.5%, metabolizable energy
3030 kcal/kg) or a diet of the same chow restricted to 50% of the intake measured by weight in paired, ad libitum fed rats. The respective diets were given from day 10 of pregnancy to term (21 days). Metyrapone (Sigma, M2696 at 0.5 mg/ml) was dissolved in drinking water and given from day 11 of gestation to term. The dose of Metyrapone was
26 based on previous studies showing effective blockade of maternal corticosterone synthesis (61). In light of multiple published reports that Metyrapone administration during gestation had no significant effects on birth weight or blood pressure in offspring of either rats (10, 41, 48) or pigs (39), a separate experimental group of control fed dams given Metyrapone was not included in the present study.
Water consumption was monitored and did not differ from controls in dams receiving Metyrapone in their drinking water. Maternal body weights and food intake were recorded daily. At postnatal day 1, all offspring from food restricted and control rat dams were cross-fostered to rat dams fed ad libitum, and litter size was culled to 4 male and 4 female per dam. Offspring were weaned at three weeks of age to ad libitum standardized laboratory chow. After weaning, the animals were then raised to 8 months of age. At this age, the animals were then transferred to Loma Linda University.
The overall study design included three groups of Sprague-Dawley rats: 1) 8- month old adults from control-fed mothers; 2) 8-month old adults from food-restricted mothers; and 3) 8-month adults from food-restricted mothers treated with Metyrapone.
Following birth, all animals were housed under a 12h:12h light-dark cycle with food and water ad libitum. At the time of study, rats were anaesthetized with 100% CO2 (1 minute) and then decapitated. The brains were rapidly removed and placed in ice cold physiological salt solution (PSS) containing (in mM): 130 NaCl, 10.0 HEPES, 6.0 glucose, 4.0 KCl, 4.0 NaHCO3, 1.8 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, and 0.025 EDTA, at pH 7.4. The main branch middle cerebral artery (MCA) was taken beginning at its origin adjacent to the Circle of Willis, cleaned of all connective tissue, and cut into lengths of 3-5 mm that were mounted on glass cannulas in an organ chamber positioned
27 on the stage of an inverted microscope (Living Systems, Burlington, VT). The proximal
cannula was connected to a pressure transducer and a windkessel reservoir of PSS, whose
pressure was controlled by a pressure servo-control system used to precisely set and change the transmural pressures of the arteries. The distal cannula was connected to a
Luer-lock valve that was opened to gently flush the lumen during the initial equilibration.
After subsequent equilibration, the outflow valve was closed and all measurements were
conducted under no-flow conditions. Arterial diameters were recorded with the SoftEdge
Acquisition Subsystem (Ion-Optix, Milton, MA). All methods used in this study have
been previously described in detail (12, 13).
Measurement of Smooth Muscle Calcium Concentration
Once cannulated and equilibrated at 21 C,̊ the artery segments were loaded with
fura-2 AM (Molecular Probes, Eugene, OR) for 20 minutes at a concentration of 1 µM as
previously described (12, 13). After loading, the arteries were washed with PSS and the
bath temperature was increased to 37 ºC. Photons emitted at 510 nm were detected at a
sampling rate of 3 Hz using an IonOptix photomultiplier system that automatically
corrected for background fluorescence. Kd values obtained from separate in vitro
calibrations were used to convert the experimental fluorescent intensity ratios (R) to
2+ [Ca2+]i over the physiological range by iterative fit to the Grynkiewicz equation: [Ca ]i
= Kd[(R-Rmin)/(Rmax- R)]Sf (24). Our calculations using this equation employed the
following averaged values: Sf (20.5), Rmin (0.3), Rmax (6.3), and Kd (251 nM).
28 Experimental Protocols
All arteries were first exposed to PSS containing 120 mM K+ for 1 to 2 minutes at 60 mmHg to verify contractility, and all arteries were denuded of endothelium by perfusing 1 mm of air through the lumen of the vessel. When contractile responses had stabilized, the 120 mM K+ was replaced with normal PSS and the arteries were re- equilibrated until baseline diameters were re-established (≈10 minutes). Next, a series of pressure steps at 20, 40, 60, 80 and 100 mmHg were applied to each artery. At each pressure, artery diameter and wall [Ca2+]i were recorded first during equilibration in normal PSS and then in PSS containing 120 mM K+. Following stabilization of the diameter and Ca2+ responses to 120 mM K+ the arteries were returned to normal PSS and the arteries were re-equilibrated until initial diameters were re-established (≈10 minutes). The pressure was then raised to the next level and the measurements were repeated. Once responses were measured at 100 mm Hg, the arteries were equilibrated in
PSS containing zero calcium with EDTA (3 mM) to determine the maximum passive diameter for each artery. This diameter was recorded at each of the previously applied pressure steps.
Fluorescent Immunohistochemistry
Arteries were fixed in 4% neutral buffered EM-grade formaldehyde (Electron
Microscopy Sciences, 15713-S) overnight, and then dehydrated, embedded in paraffin, and cut into sections 5 µm thick. The sections were then deparaffinized, rehydrated, immersed in citrate buffer at pH 6.03, and microwaved for 5 minutes to facilitate antigen recovery. The sections were then incubated in 1% Bovine Serum Albumin (BSA, Santa
29 Cruz Biotechnology, SC-2323) with Triton X-100 (Sigma-Aldrich, T-8787) blocking buffer for an hour. Next, the sections were double-stained with primary antibodies reactive with Smooth Muscle α-Actin (Sigma-Aldrich, A5228 @ 1:200), SM2 Myosin
(Abcam, Ab53219 @ 1:500), SM1 Myosin (Abcam, Ab681 @ 1:100), and SMemb
Myosin (Covance, PRB-445P @ 1:400), applied and incubated at 4 ºC overnight. The following day, the sections were washed in PBS then equilibrated in darkness for two hours at room temperature with two secondary antibodies labeled with Dylight-488 and
Dylight-649, and then examined with our Olympus FV1000 at an optical section thickness of 1 μm, a lateral resolution of 2 nm, and a numerical aperture of 18. The degree of colocalization between the two markers was determined using the
Colocalization Coefficient, because this measure was independent of absolute marker intensities. For the colocalization analysis, coronal sections were extracted from the parent images using a masking routine to eliminate background contributions to the measurement statistics. These methods have previously been described in detail (12, 13).
Chemical and Reagents
All chemicals were purchased from Sigma (St. Louis, MO), except pluronic acid and fura-2 AM which were acquired from Molecular Probes / Invitrogen.
Data Analysis and Statistics
All values are given in the text as means ± SE, and statistical significance implies
P < 0.05 unless stated otherwise. In all cases, n refers to the number of animals studied.
Values of artery diameter, and wall calcium were analyzed via two-way ANOVA with
30 repeated measures using age (pup vs. adults) and pressure as factors. Post hoc
comparisons were performed using the Fisher Paired Least Significant Difference
analysis. Values for the Colocalization Coefficient were compared using a Behren’s-
Fischer analysis with pooled variance. For analysis of myosin isoform expression, all
image pixels with greater than median intensity for α-actin were segregated into two
groups with intensities either above or below the median intensity for each myosin
isoform, respectively (SM2 and SMemb). The percentages of total pixels in each of these
groups was calculated for each age group, and then compared between age groups using a
Behren’s-Fischer analysis with pooled variance. All data sets were normally distributed,
as verified using a D’Agostino-Pearson K2 test, and homogeneity of variance within
ANOVA was verified using a Bartlett’s-Cochran test (68).
Results
This study is based on data obtained from 15 rats: five were 8-month old adults from control-fed mothers; five were 8-month old adults from food-restricted mothers; and five were 8-month adults from food-restricted mothers treated with Metyrapone; all were cross-fostered from different dams to minimize any genetic or litter-specific effects. One artery segment was used from each animal for contractility studies, and the contralateral artery was taken for immunohistochemistry and confocal imaging.
31 Effects of Changes in Pressure on Maximum Passive Diameter in Control, MFR and
MFR +Metyrapone MCAs
In Control arteries treated with EGTA to eliminate active tone, average diameters increased progressively from 220 ± 13 µm at 20 mm Hg to 296 ± 10 µm at 100 mm Hg
(Figure 1, upper panel). The relation between pressure and diameter was shifted upward
in the MFR group, in which diameters averaged 243 ± 7 µm at 20 mm Hg to 299 ± 11 µm
at 100 mm Hg. The MFR values were significantly greater than observed in Controls at
both 20 and 40 mm Hg. Treatment with Metyrapone yielded a pressure-diameter relation
that was not significantly different than that in Controls at any pressure; diameters
averaged 228 ± 5 µm at 20 mm Hg and 299 ± 5 µm at 100 mm Hg. At 20 mm Hg, the
diameter in the Metyrapone group was significantly less than that in the MFR group.
Calculations of incremental compliance from the slopes of the pressure-diameter
relations revealed that in Control group arteries, compliance decreased progressively
from 1.29 ± 0.25 µm/mm Hg at 30 mm Hg to 0.66 ± 0.02 µm/mm Hg at 90 mm Hg
(Figure 1, lower panel). The relation between compliance and pressure was significantly
shifted downward in the MFR group such that compliance averaged 0.92 ± 0.12 µm/mm
Hg at 30 mm Hg to 0.55 ± 0.0 µm/mm Hg at 90 mm Hg. Treatment with Metyrapone
yielded a pressure-compliance relation that was not significantly different than that in
Controls at any pressure. In the Metyrapone group, compliance values averaged 1.22 ±
0.20 at 30 mm Hg and 0.56 ± 0.09 at 90 mm Hg.
32
Figure 1: Effects of Maternal Food Restriction on Passive Arterial Diameter and Compliance. Upper Panel: Passive artery diameters were determined in the presence of 3 mM EGTA, which inhibited all active tension. Compared to arteries from the Control group animals, arteries in the MFR group had larger diameters at all pressures, and these differences were significant at 20 and 40 mm Hg (P<0.05, *). Artery diameters in the Metyrapone group were not significantly different than those in the Control group at any pressure and were significantly less than in the MFR group at 20 mm Hg (P<0.05, #). Lower Panel: Calculations of incremental compliance from the slopes of the pressure- diameter relations indicated that MFR significantly decreased compliance relative to Control and this effect was significant at 30 and 70 mm Hg (P<0.05, *). Values in the MFR+Metyrapone group did not differ significantly from those in controls at any pressure. In both panels, error bars indicate SEM for N=5 at each point.
33 Effects of Changes in Pressure on Myogenic Contractility in Control, MFR and MFR
+Metyrapone MCAs
Stretch-induced active (myogenic) contractions were calculated at each transmural
pressure as differences in artery diameter measured in PSS and EGTA solutions. In
Control arteries, these ∆D values increased from 36 ± 10 µm at 20 mm Hg to a peak
value of 75 ± 7 µm at 60 mm Hg. At higher pressures, the ∆D values decreased to 55 ± 7
µm at 100 mm Hg (Figure 2, upper panel). The ∆D values in arteries from the MFR
group were generally less than observed in the Control group, and these differences were
significant at 40 and 60 mm Hg. In the MFR+Metyrapone group, the ∆D values were
significantly less than observed in the Control group at 60 mm Hg but were greater than
observed in Controls at 100 mm Hg.
Stretch-induced changes in cytosolic calcium concentration were calculated at each
transmural pressure as differences in concentration measured in PSS and EGTA
solutions. In Control arteries, these ∆[Ca2+] values increased from 96 ± 3 nM at 20 mm
Hg to a peak value of 141 ± 7 nM at 80 mm Hg (Figure 2, middle panel). The ∆[Ca2+]
values in arteries from the MFR group were not significantly different than observed in
the Control group at any pressure. In contrast, the ∆[Ca2+] values observed in the
MFR+Metyrapone group were significantly greater than observed in Control arteries at
all pressure, and were significantly greater than observed in MFR arteries at 20, 60, 80
and 100 mm Hg.
Myofilament calcium sensitivity was estimated as the ratio between the stretch-
induced ∆D response and the ∆[Ca2+] response at each pressure. The values of this ratio were significantly less in the MFR group than in the Control group at all pressures except
34 100 mm Hg (Figure 2, bottom panel). Values of this ratio in the MFR+Metyrapone group were also significantly less than in the Control group, and at all pressures.
35
Figure 2. Effects of MFR and Metyrapone on Myogenic Pressure-Diameter Relations. Shown in the upper panel are stretch-induced (myogenic) changes in diameter measured in each of the experimental groups. The stretch-induced ∆D responses were generally less in the MFR group than in controls, and these differences were significant at 40 and 60 mm Hg (P<0.05, *). In the MFR+Metyrapone group, the ∆D values were significantly less than observed in the Control group at 60 mm Hg, but were greater than observed in Controls at 100 mm Hg. As indicated in the middle panel, stretch-induced changes in cytosolic calcium were not different in arteries from the Control and MFR groups. In contrast, the stretch-induced ∆[Ca2+] values observed in the MFR+Metyrapone group were significantly greater than seen in Control arteries at all pressures (P<0.05, *), and were significantly greater than observed in MFR arteries at 20, 60, 80 and 100 mm Hg (P<0.05, #). Shown in the lower panel are estimates of myofilament calcium sensitivity, calculated as the ratio between the stretch-induced ∆D response and the ∆[Ca2+] response at each pressure. The values of this ratio were significantly less in the MFR group than in the Control group at all pressures except 100 mm Hg. Values of this ratio in the MFR+Metyrapone group were also significantly less than in the Control group, and at all pressures. Error bars indicate SEM for N=5 at each point.
36 Effects of Changes in Pressure on Potassium-Induced Contractility in Control, MFR
and MFR +Metyrapone MCAs
Potassium-induced active contractions were calculated at each transmural pressure
as differences in artery diameter measured in high potassium (120 mM) and PSS
solutions. In Control arteries, these ∆D values did not vary with pressure and ranged
from a minimum of 36 ± 7 µm at 100 mm Hg to a maximum of 62 ± 9 µm at 40 mm Hg
(Figure 3, upper panel). Potassium-induced ∆D values in arteries from the MFR group
were significantly greater than control at 40 mm Hg, but were significantly less than
control at 80 mm Hg. The ∆D values in the MFR+Metyrapone group were significantly
greater than in the Control group only at 100 mm Hg, but were significantly greater than
in the MFR+Metyrapone arteries at both 80 and 100 mm Hg.
Potassium-induced changes in cytosolic calcium concentration were calculated at
each transmural pressure as differences in concentration measured in high potassium and
PSS solutions. In Control arteries, these ∆[Ca2+] values ranged from a maximum of 211
± 11 nM at 20 mm Hg to a minimum of 162 ± 18 nM at 80 mm Hg (Figure 3, middle panel). At all pressures, ∆[Ca2+] values were significantly greater in the MFR group than
in the Control group and ranged from 302 ± 30 nM at 20 mm Hg to 230 ± 31 nM at 80
mm Hg. In the MFR+Metyrapone group, ∆[Ca2+] values were also significantly greater
than in the Control group at all pressures, but did not differ significantly from values in
the MFR group.
Potassium-induced changes in myofilament calcium sensitivity were estimated as
the ratio between the K+-induced ∆D response and the ∆[Ca2+] response at each pressure.
Values of this ratio were significantly less in the MFR group than in the Control group at
37 20 and 80 mm Hg (Figure 3, bottom panel). Values of this ratio in the
MFR+Metyrapone group were significantly less than in the Control group at 20, 40 and
80 mm Hg, but were significantly greater than in the Control group at 100 mm Hg.
Values of this ratio in the MFR+Metyrapone group were also significantly less than in the MFR group at 20 and 40 mm Hg.
38
Figure 3. Effects of Changes in Pressure on Potassium-Induced Contractility in Control, MFR, and MFR+Metyrapone MCA’s. Shown in the upper panel are stretch-induced (myogenic) changes in diameter. In arteries from the MFR group, the ∆D were significantly greater than Control at 40 mm Hg, but were significantly less than control at 80 mm Hg (P<0.05, *). In the MFR+Metyrapone arteries, ∆D values were significantly greater than in the Control group at 100 mm Hg (P<0.05, *) and were greater than in the MFR+Metyrapone at both 80 and 100 mm Hg (P<0.05, #). Shown in the middle panel are potassium-induced changes in cytosolic calcium, which were significantly greater in both the MFR and MFR+Metyrapone groups (P<0.05, *) than in the Control group, at all pressures. Values of ∆[Ca2+] in the MFR and MFR+Metyrapone groups did not differ significantly from one another at any pressure. Shown in the lower panel are estimates of myofilament calcium sensitivity (∆D/∆[Ca2+]), which were significantly less in the MFR group than in the Control group at 20 and 80 mm Hg (P<0.05, *). Values of this ratio in the MFR+Metyrapone group were significantly less than in the Control group at 20, 40 and 80 mm Hg, but were significantly greater than in the Control group at 100 mm Hg. Values of this ratio in the MFR+Metyrapone group were also significantly less than in the MFR group at 20 and 40 mm Hg (P<0.05, #). Error bars indicate SEM for N=5 at each point.
39 Effects of Maternal Food Restriction and Metyrapone on Contractile Protein
Colocalization and Smooth Muscle in MCAs
To assess the effects of MFR with or without Metyrapone on smooth muscle phenotype, concentric rings of rat middle cerebral arteries were immunostained to detect colocalization of Smooth Muscle α-Actin with the Non-Muscle, SM1, or SM2 isoforms of myosin heavy chain (Figure 4). Colocalization of Smooth Muscle α-Actin with the
Non-Muscle isoform was significantly greater in the MFR group than in either the
Control group or the MFR+Metyrapone group (Figure 5). Colocalization of Smooth
Muscle α-Actin with the SM1 isoform was also significantly greater in the MFR group than in the Control group, but the SM1 colocalization value in the MFR+Metyrapone did not differ significantly from either the Control or MFR groups. For colocalization of
Smooth Muscle α-Actin with the SM2 isoform, the values in the Control and MFR groups were similar, but the value in the MFR+Metyrapone group was significantly greater than in either the Control or MFR groups.
40
Figure 4. Effects of Maternal Food Restriction and Metyrapone on Contractile Protein Colocalization and Smooth Muscle in MCA’s, Composite. Concentric rings of rat middle cerebral arteries were fixed in 4% paraformaldehyde, cut at 5 µm, and immunostained to quantify colocalization of Smooth Muscle α-Actin with one of three myosin heavy chain isoforms. Shown here are representative sections from all three experimental groups immunostained for colocalization of Smooth Muscle α-Actin with the Non-Muscle isoform of myosin heavy chain.
41
Figure 5. Effects of Maternal Food Restriction and Metyrapone on Contractile Protein Colocalization and Smooth Muscle in MCA’s, Quantitative Analysis. Colocalization of Smooth Muscle α-Actin with the myosin heavy chain isoforms was quantified to assess the effects of MFR and Metyrapone on the fraction of smooth muscle exhibiting immature (Non-Muscle), intermediate (SM1), or fully differentiated (SM2) contractile smooth muscle phenotypes. Colocalization with Smooth Muscle α-Actin was with Non-Muscle myosin heavy chain was significantly greater in the MFR group than in either the Control group or the MFR+Metyrapone group (Figure 5). Colocalization of Smooth Muscle α- Actin with the SM1 isoform was also significantly greater in the MFR group than in the Control group, but the SM1 colocalization value in the MFR+Metyrapone did not differ significantly from either the Control or MFR groups. For colocalization of Smooth Muscle α-Actin with the SM2 isoform, the values in the Control and MFR groups were similar, but the value in the MFR+Metyrapone group was significantly greater than in either the Control or MFR groups. Error bars indicate SEM for N=5 for each mean.
42 Discussion
The present study explores the hypothesis that glucocorticoids contribute to the effects of maternal food restriction (MFR) on structure-function relations in the cerebral circulation of adult offspring. In middle cerebral arteries (MCA), MFR reduced incremental compliance and this effect was largely prevented by treatment with
Metyrapone, a corticosterone synthesis inhibitor. Myogenic reactivity was attenuated by
MFR, largely through inhibition of myofilament calcium sensitivity, and this inhibition was resistant to Metyrapone. MFR had mixed effects on potassium-induced contractions that included enhancement of the calcium response to potassium together with inhibition of myofilament calcium sensitivity. The effects of MFR on potassium-induced tone were minimally altered by Metyrapone. Colocalization of Non-Muscle Myosin Heavy Chain
(NM-MHC) with smooth muscle α-actin, an index of the proportion of functionally immature smooth muscle in the artery, was enhanced by MFR and this effect was reduced by Metyrapone. In contrast, colocalization of SM2 Myosin Heavy Chain (SM-
MHC) with smooth muscle α-actin, an index of the proportion of fully differentiated contractile smooth muscle, was unaffected by MFR but was enhanced by Metyrapone.
Together, these results suggest that MFR selectively alters multiple aspects of structure- function relations in cerebral arteries, and that only some of these effects involve the actions of glucocorticoids.
One of the most well documented effects of glucocorticoids on arterial structure is their ability to increase stiffness (34). Given the capacity of MFR to increase maternal glucocorticoids (43), our first series of experiments examined if MFR could increase cerebral artery stiffness and thereby reduce passive compliance. These measurements
43 revealed that MFR reduced passive compliance and, more importantly, that treatment with Metyrapone virtually eliminated this effect (Figure 1). Given the key roles of elastin and collagen as determinants of arterial stiffness (60, 64), these findings suggest that
MFR reduces cerebrovascular compliance through changes in matrix protein abundance and organization, mediated by increased activity of corticosterone on the glucocorticoid receptor (6, 42) and possibly also on the mineralocorticoid receptor (46). These results are also consistent with possible effects of MFR on matrix metalloproteinase (MMP) activities or tissue inhibitors of metalloproteinases (TIMP), as suggested by other studies
(21, 27, 37). Although the precise mechanisms remain unclear, the results suggest that
MFR can have long-term, “programming” effects on cerebrovascular compliance in a glucocorticoid-dependent manner.
Compared to their effects on vascular structure and compliance, the long-term effects of glucocorticoids on vascular function are more poorly understood, due largely to their highly artery and tissue-specific effects (22, 25, 67). To evaluate if MFR might produce long-term influences on cerebrovascular contractility through a glucocorticoid dependent pathway, our second series of experiments assayed pressure-dependent myogenic contractility in cerebral arteries isolated from adult offspring of food-restricted dams. In these experiments, MFR depressed myogenic contractility (Figure 2, upper panel), but had very little effect on the relation between transmural pressure and cytosolic calcium concentration (Figure 2, middle panel). Because contractile tone is the product of cytosolic calcium concentration and myofilament calcium sensitivity (58), the observed pattern suggests that the long-term effects of MFR on myogenic contractility were attributable largely to decreased myofilament calcium sensitivity. Consistent with
44 this interpretation, MFR decreased the ratio between artery diameter and cytosolic
calcium (Figure 2, bottom panel), an index of myofilament calcium sensitivity (12).
Regarding the role of glucocorticoids in these “programming” effects of MFR,
Metyrapone had very little influence on MFR-induced depression of myogenic contractility (Figure 2, upper panel), but interfered significantly with regulation of cytosolic calcium (Figure 2, middle panel). This latter finding suggests that glucocorticoids are essential for calcium homeostasis in cerebral arteries, at least in adult offspring of food-restricted dams. Although glucocorticoids can depress contractility in cerebral arteries (19), and can inhibit myofilament sensitivity in airway smooth muscle
(23) and uterine arteries (66), Metyrapone had very little effect on myofilament calcium sensitivity in cerebral arteries of MFR offspring (Figure 2, bottom panel). Altogether,
these findings suggest that MFR attenuates cerebrovascular myogenic contractility
through long-term depression of myofilament calcium sensitivity via mechanisms largely
independent of glucocorticoids.
Another important characteristic of vascular function is maximum contractile capacity, which can be elucidated by exposure to high concentrations of potassium that depolarize the smooth muscle membrane, enable the influx of extracellular calcium, and maximally stimulate the contractile apparatus in a receptor-independent manner (32). In our experiments, MFR produced long-term effects on contractile responses to potassium depolarization in a pressure-dependent manner (Figure 3) that were quite distinct from the pattern observed for myogenic contractility (Figure 2). Compared to control responses, MFR responses to potassium depolarization were elevated at 40 mm Hg, but depressed at 80 mm Hg, suggesting that the fundamental stress-strain relationship in
45 these arteries underwent long-term alteration by MFR. These results at lower transmural pressures (20 – 60 mm Hg) generally agreed with the findings of Ozaki et al. (55), who
reported that MFR enhanced potassium-induced contractility of isolated femoral arteries.
In turn, the inconsistencies between the present results at higher pressures and the Ozaki findings could be due to differences in artery type (femoral vs. cerebral), method of contractile measurement (pressurized myography vs. wire-mount isometric myography),
or food-restriction model (70% MFR for 0-18 days gestation vs. 50% MFR for 11-21
days gestation).
Unlike the Ozaki study, however, the present experiments examined the sustained
effects of MFR on calcium regulation and revealed that MFR increased cytosolic calcium
concentrations at all pressures, suggesting that the long-term effects of MFR on overall
contractility were not dependent primarily upon pressure-dependent changes in calcium
handling (Figure 3, middle panel). Instead, the data suggest that MFR increased either
release or influx of calcium or, alternatively, decreased extrusion or sequestration of
calcium in these arteries (45). The contractile effects of the elevated calcium levels in the
arteries from the MFR offspring were partially offset by parallel depression of
myofilament calcium sensitivity between 20 and 80 mm Hg (Figure 3, lower panel). The
ability of MFR to modulate myofilament sensitivity further suggests that MFR either
decreased the ratio between Myosin Light Chain Kinase and Myosin Light Chain
Phosphatase activities (thick filament regulation), and/or depressed the ability of
activated myosin heavy chain to generate force through cross-bridge interactions with
actin (thin filament regulation) (53). Altogether, these results suggest that MFR can exert
long-term effects on either calcium regulation through possible alterations in the function
46 of key proteins such as L-channels, PMCA, or SERCA, or alternatively may modulate
abundances and activities of proteins governing myofilament calcium sensitivity such as
MLCK, MLCP, CPI-17, Rho-Kinase, caldesmon, HSP27, etc. (3, 11).
One of the possible mechanisms through which MFR could modulate multiple key vascular proteins is through activation of glucocorticoid receptors (19, 40, 49). Consistent with a role for glucocorticoids in MFR-induced changes in contractility, Metyrapone blocked the effects of MFR on potassium contractions at low (40 mm Hg) and high (80 mm Hg) pressures (Figure 3, upper panel). Despite these effects of Metyrapone on
potassium-induced contractility, Metyrapone had no significant effect on cytosolic
calcium, suggesting that glucocorticoids are not involved in the programmed effects of
MFR on calcium handling during potassium contractions (Figure 3, middle panel). This
result agrees with previous findings in cardiac myocytes that L-channel density is
increased, and that SERCA pump abundance is decreased by glucocorticoids (15),
leading to increased cytosolic calcium concentrations. In contrast, Metyrapone further
depressed myofilament calcium sensitivity at low pressures (20 and 40 mm Hg), but
increased it at high pressure (100 mm Hg) in potassium-contracted arteries, suggesting
that glucocorticoids probably contribute little to the long-term effects of MFR on calcium
sensitivity (Figure 3, lower panel). In combination, these results emphasize that MFR
influences calcium handling and calcium sensitivity very differently in stretch-induced
and potassium-induced contractions through mechanisms that are largely independent of
the sustained effects of glucocorticoids.
Another general mechanism through which MFR could alter cerebrovascular
contractility is by modulation of the phenotype of smooth muscle (54) in cerebral arteries.
47 A transition from a contractile to a non-contractile smooth muscle phenotype can
dramatically alter calcium handling, contractility and structure (28, 65), and such
dedifferentiation can be identified through changes in patterns of contractile protein
expression and colocalization (1, 12, 30). From this perspective, MFR produced long-
term increases in the fraction of smooth muscle α-actin colocalized with Non-Muscle
Myosin Heavy Chain (NM-MHC)(Figure 4), suggesting an increase in the fraction of
non-contractile smooth muscle (20, 54). This result suggests that MFR could promote
either inward migration of progenitor smooth muscle cells, or dedifferentiation and/or
proliferation of pre-existing contractile smooth muscle (56). Arguing against
dedifferentiation, which would be expected to decrease the number of contractile smooth
muscle cells, MFR had no effect on the colocalization of SM2 MHC with smooth muscle
α-actin, suggesting that the proportion of contractile smooth muscle cells was unchanged
(Figure 5). In addition, the colocalization of SM1 MHC with smooth muscle α-actin,
which is an indicator of partially differentiated contractile smooth muscle (20), was
increased by MFR. Together, these findings suggest that MFR produced long-term increases in the total fractions of smooth muscle cells in both the contractile and non-
contractile phenotypes, which could be explained only by proliferation of existing smooth
muscle, or inward migration and/or differentiation of smooth muscle progenitors in the
artery wall (54).
With respect to the involvement of glucocorticoids in the effects of MFR on smooth
muscle phenotype, treatment with Metyrapone significantly decreased colocalization of
NM-MHC with smooth muscle α-actin (Figure 5). This finding suggests that
glucocorticoids contributed to the MFR-induced increases in colocalization of NM-MHC
48 with smooth muscle α-actin, which again could indicate inward migration of progenitor smooth muscle cells, dedifferentiation, or smooth muscle proliferation (56). Whereas
Metyrapone had minimal effects on SM1 colocalization, it significantly enhanced colocalization of SM2 MHC with smooth muscle α-actin, which supports the view that glucocorticoids promote contractile dedifferentiation in response to MFR. This interpretation is in contrast with previous reports that antenatal dexamethasone treatment can accelerate short-term vascular differentiation (26), and emphasizes that short-term and long-term effects of glucocorticoids are not always equivalent. On the other hand, the present results agree with previous reports that glucocorticoids promote proliferation in vascular smooth muscle cells (63), and thereby could contribute to the long-term effects of MFR on smooth muscle phenotype. These changes in smooth muscle phenotype, in turn, could help explain the complex effects of MFR on calcium handling, myofilament calcium sensitivity, and overall contractility.
Overall, the present experiments support the hypothesis that early maternal food restriction fundamentally alters long-term structure-function relationships in the middle cerebral arteries of adult offspring, as revealed by pressure myography and confocal microscopy. MFR significantly decreased compliance in offspring cerebral arteries, and this effect was rescued by treatment with the corticosterone synthesis inhibitor
Metyrapone, suggesting that this effect of MFR was mediated by increased glucocorticoid exposure during gestation. MFR also caused long-term depression of myogenic reactivity due largely to attenuation of myofilament calcium sensitivity through mechanisms that were resistant to Metyrapone, and thus appeared independent of glucocorticoids. Similarly, MFR programmed potassium-induced contractions but in a
49 manner distinct from its effects on myogenic contractility; potassium contractions were enhanced at low pressure, and depressed at high pressure, by MFR. During potassium contractions, MFR increased cytosolic calcium at all pressures through mechanisms resistant to Metyrapone, but MFR generally depressed myofilament calcium sensitivity through mechanisms that were enhanced by Metyrapone at low pressures, but antagonized at high pressures. In relation to smooth muscle phenotype, MFR gave rise to enhanced proportions of cells in a non-contractile phenotype without diminishing the proportion of fully differentiated contractile cells, suggesting either a long-term shift towards an increased proliferation or inward migration of smooth muscle progenitors.
Metyrapone generally antagonized the effects of MFR on smooth muscle phenotype, suggesting that glucocorticoids contribute to changes in smooth muscle phenotype in both the long term as well as the short term. Owing to the strong relationships among smooth muscle phenotype, contractility, and structure, the sustained influences of MFR on smooth muscle phenotype help explain both the glucocorticoid-dependent and – independent general effects of MFR programming on cerebrovascular structure and function. These findings tempt speculation that MFR might increase vulnerability to stroke and other cerebrovascular diseases, in parallel with its ability to increase the risk of cardiac disease in adult offspring of food-restricted mothers (7, 8).
Acknowledgements
The authors greatly appreciate the contribution from Shelton M. Charles who performed the myography experiments. The authors are also grateful for the excellent technical assistance of Jenna M Abrassart, who performed the confocal preparations and
50 measurements. The work reported in this manuscript was supported by National
Institutes of Health Grants HD-054920, HD-31266, HL-54120, HL-64867, and the Loma
Linda University School of Medicine.
51 References
1. Adeoye OO, Butler SM, Hubbell MC, Semotiuk A, Williams JM, and Pearce WJ. Contribution of increased VEGF receptors to hypoxic changes in fetal ovine carotid artery contractile proteins. Am J Physiol Cell Physiol 304: C656-665, 2013.
2. Ahmed MA. Impact of vitamin D(3) on cardiovascular responses to glucocorticoid excess. Journal of physiology and biochemistry 2012.
3. Akata T. Cellular and molecular mechanisms regulating vascular tone. Part 2: regulatory mechanisms modulating Ca2+ mobilization and/or myofilament Ca2+ sensitivity in vascular smooth muscle cells. Journal of anesthesia 21: 232-242, 2007.
4. Almond K, Bikker P, Lomax M, Symonds ME, and Mostyn A. The influence of maternal protein nutrition on offspring development and metabolism: the role of glucocorticoids. Proc Nutr Soc 71: 198-203, 2012.
5. Atinmo T, Pond WG, and Barnes RH. Effect of maternal energy vs. protein restriction on growth and development of progeny in swine. J Anim Sci 39: 703-711, 1974.
6. Ayari H, Legedz L, Lantelme P, Feugier P, Randon J, Cerutti C, Lohez O, Scoazec JY, Li JY, Gharbi-Chihi J, and Bricca G. Auto-amplification of cortisol actions in human carotid atheroma is linked to arterial remodeling and stroke. Fundamental & clinical pharmacology 28: 53-64, 2014.
7. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, and Robinson JS. Fetal nutrition and cardiovascular disease in adult life. Lancet 341: 938-941, 1993.
8. Barker DJ, Osmond C, Golding J, Kuh D, and Wadsworth ME. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. BMJ 298: 564-567, 1989.
9. Bertram C, Khan O, Ohri S, Phillips DI, Matthews SG, and Hanson MA. Transgenerational effects of prenatal nutrient restriction on cardiovascular and hypothalamic-pituitary-adrenal function. J Physiol 586: 2217-2229, 2008.
10. Bogdarina I, Haase A, Langley-Evans S, and Clark AJ. Glucocorticoid effects on the programming of AT1b angiotensin receptor gene methylation and expression in the rat. PloS one 5: e9237, 2010.
11. Brini M, and Carafoli E. The plasma membrane Ca(2)+ ATPase and the plasma membrane sodium calcium exchanger cooperate in the regulation of cell calcium. Cold Spring Harbor perspectives in biology 3: 2011.
12. Charles SM, Zhang L, Cipolla MJ, Buchholz JN, and Pearce WJ. Roles of cytosolic Ca2+ concentration and myofilament Ca2+ sensitization in age-dependent
52 cerebrovascular myogenic tone. Am J Physiol Heart Circ Physiol 299: H1034-1044, 2010.
13. Charles SM, Zhang L, Longo LD, Buchholz JN, and Pearce WJ. Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases in Ca2+ in rat cerebral arteries. Am J Physiol Regul Integr Comp Physiol 293: R737- 744, 2007.
14. Cottrell EC, Holmes MC, Livingstone DE, Kenyon CJ, and Seckl JR. Reconciling the nutritional and glucocorticoid hypotheses of fetal programming. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 26: 1866-1874, 2012.
15. De P, Roy SG, Kar D, and Bandyopadhyay A. Excess of glucocorticoid induces myocardial remodeling and alteration of calcium signaling in cardiomyocytes. J Endocrinol 209: 105-114, 2011.
16. Desai M, Babu J, and Ross MG. Programmed metabolic syndrome: prenatal undernutrition and postweaning overnutrition. Am J Physiol Regul Integr Comp Physiol 293: R2306-2314, 2007.
17. Desai M, Crowther NJ, Lucas A, and Hales CN. Organ-selective growth in the offspring of protein-restricted mothers. Br J Nutr 76: 591-603, 1996.
18. Desai M, Gayle D, Babu J, and Ross MG. Permanent reduction in heart and kidney organ growth in offspring of undernourished rat dams. Am J Obstet Gynecol 193: 1224-1232, 2005.
19. Eckman DM, Kerr BA, Fuloria M, Simandle SA, Watt SE, Rose JC, and Figueroa JP. Antenatal betamethasone alters vascular reactivity in adult female ovine cerebral arteries. Pediatr Res 68: 344-348, 2010.
20. Eddinger TJ, and Meer DP. Myosin II isoforms in smooth muscle: heterogeneity and function. Am J Physiol Cell Physiol 293: C493-508, 2007.
21. Forster C, Kahles T, Kietz S, and Drenckhahn D. Dexamethasone induces the expression of metalloproteinase inhibitor TIMP-1 in the murine cerebral vascular endothelial cell line cEND. J Physiol 580: 937-949, 2007.
22. Goldsmith AM, Hershenson MB, Wolbert MP, and Bentley JK. Regulation of airway smooth muscle alpha-actin expression by glucocorticoids. American journal of physiology Lung cellular and molecular physiology 292: L99-L106, 2007.
23. Goto K, Chiba Y, Sakai H, and Misawa M. Glucocorticoids inhibited airway hyperresponsiveness through downregulation of CPI-17 in bronchial smooth muscle. European journal of pharmacology 591: 231-236, 2008.
53 24. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. The Journal of biological chemistry 260: 3440-3450, 1985.
25. Hadoke PW, Lindsay RS, Seckl JR, Walker BR, and Kenyon CJ. Altered vascular contractility in adult female rats with hypertension programmed by prenatal glucocorticoid exposure. J Endocrinol 188: 435-442, 2006.
26. Hai CM, Sadowska G, Francois L, and Stonestreet BS. Maternal dexamethasone treatment alters myosin isoform expression and contractile dynamics in fetal arteries. Am J Physiol Heart Circ Physiol 283: H1743-1749, 2002.
27. Hartmann C, El-Gindi J, Lohmann C, Lischper M, Zeni P, and Galla HJ. TIMP-3: a novel target for glucocorticoid signaling at the blood-brain barrier. Biochem Biophys Res Commun 390: 182-186, 2009.
28. House SJ, Potier M, Bisaillon J, Singer HA, and Trebak M. The non-excitable smooth muscle: calcium signaling and phenotypic switching during vascular disease. Pflugers Archiv : European journal of physiology 456: 769-785, 2008.
29. Hsueh AM, Simonson M, Chow BF, and Hanson HM. The importance of the period of dietary restriction of the dam on behavior and growth in the rat. J Nutr 104: 37-46, 1974.
30. Hubbell MC, Semotiuk AJ, Thorpe RB, Adeoye OO, Butler SM, Williams JM, Khorram O, and Pearce WJ. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries. Am J Physiol Cell Physiol 303: C1090-1103, 2012.
31. Ivers LC, and Cullen KA. Food insecurity: special considerations for women. Am J Clin Nutr 94: 1740S-1744S, 2011.
32. Jackson WF. Ion channels and vascular tone. Hypertension 35: 173-178, 2000.
33. Kawamura M, Itoh H, Yura S, Mogami H, Suga S, Makino H, Miyamoto Y, Yoshimasa Y, Sagawa N, and Fujii S. Undernutrition in utero augments systolic blood pressure and cardiac remodeling in adult mouse offspring: possible involvement of local cardiac angiotensin system in developmental origins of cardiovascular disease. Endocrinology 148: 1218-1225, 2007.
34. Kelly BA, Lewandowski AJ, Worton SA, Davis EF, Lazdam M, Francis J, Neubauer S, Lucas A, Singhal A, and Leeson P. Antenatal glucocorticoid exposure and long-term alterations in aortic function and glucose metabolism. Pediatrics 129: e1282-1290, 2012.
35. Khorram O, Han G, Bagherpour R, Magee TR, Desai M, Ross MG, Chaudhri AA, Toloubeydokhti T, and Pearce WJ. The Effect of Maternal Undernutrition On
54 Vascular Expression of Micro and Messenger RNA in Newborn and Aging Offspring. Am J Physiol Regul Integr Comp Physiol 298: R1366-1374, 2010.
36. Khorram O, Khorram N, Momeni M, Han G, Halem J, Desai M, and Ross MG. Maternal undernutrition inhibits angiogenesis in the offspring: a potential mechanism of programmed hypertension. Am J Physiol Regul Integr Comp Physiol 293: R745-753, 2007.
37. Khorram O, Momeni M, Desai M, and Ross MG. Nutrient restriction in utero induces remodeling of the vascular extracellular matrix in rat offspring. Reprod Sci 14: 73-80, 2007.
38. Khorram O, Momeni M, Ferrini M, Desai M, and Ross MG. In utero undernutrition in rats induces increased vascular smooth muscle content in the offspring. Am J Obstet Gynecol 196: 486 e481-488, 2007.
39. Klemcke HG, Vallet JL, and Christenson RK. Lack of effect of metyrapone and exogenous cortisol on early porcine conceptus development. Exp Physiol 91: 521- 530, 2006.
40. Langley-Evans SC. Fetal programming of cardiovascular function through exposure to maternal undernutrition. Proc Nutr Soc 60: 505-513, 2001.
41. Langley-Evans SC. Intrauterine programming of hypertension in the rat: nutrient interactions. Comp Biochem Physiol A Physiol 114: 327-333, 1996.
42. Leitman DC, Benson SC, and Johnson LK. Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells. The Journal of cell biology 98: 541-549, 1984.
43. Lesage J, Blondeau B, Grino M, Breant B, and Dupouy JP. Maternal undernutrition during late gestation induces fetal overexposure to glucocorticoids and intrauterine growth retardation, and disturbs the hypothalamo-pituitary adrenal axis in the newborn rat. Endocrinology 142: 1692-1702, 2001.
44. Lingas R, Dean F, and Matthews SG. Maternal nutrient restriction (48 h) modifies brain corticosteroid receptor expression and endocrine function in the fetal guinea pig. Brain Res 846: 236-242, 1999.
45. Marin J, Encabo A, Briones A, Garcia-Cohen EC, and Alonso MJ. Mechanisms involved in the cellular calcium homeostasis in vascular smooth muscle: calcium pumps. Life Sci 64: 279-303, 1999.
46. McCurley A, McGraw A, Pruthi D, and Jaffe IZ. Smooth muscle cell mineralocorticoid receptors: role in vascular function and contribution to cardiovascular disease. Pflugers Archiv : European journal of physiology 2013.
55 47. McMillen IC, and Robinson JS. Developmental origins of the metabolic syndrome: prediction, plasticity, and programming. Physiological reviews 85: 571-633, 2005.
48. McMullen S, and Langley-Evans SC. Maternal low-protein diet in rat pregnancy programs blood pressure through sex-specific mechanisms. Am J Physiol Regul Integr Comp Physiol 288: R85-90, 2005.
49. Meaney MJ, Szyf M, and Seckl JR. Epigenetic mechanisms of perinatal programming of hypothalamic-pituitary-adrenal function and health. Trends in molecular medicine 13: 269-277, 2007.
50. Mesquita FF, Gontijo JA, and Boer PA. Expression of renin-angiotensin system signalling compounds in maternal protein-restricted rats: effect on renal sodium excretion and blood pressure. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 25: 380-388, 2010.
51. Mitchell NS, Catenacci VA, Wyatt HR, and Hill JO. Obesity: overview of an epidemic. The Psychiatric clinics of North America 34: 717-732, 2011.
52. Nascimento FA, Ceciliano TC, Aguila MB, and Mandarim-de-Lacerda CA. Maternal vitamin D deficiency delays glomerular maturity in F1 and F2 offspring. PloS one 7: e41740, 2012.
53. Nauli SM, Williams JM, Gerthoffer WT, and Pearce WJ. Chronic hypoxia modulates relations among calcium, myosin light chain phosphorylation, and force differently in fetal and adult ovine basilar arteries. Journal of applied physiology (Bethesda, Md : 1985) 99: 120-127, 2005.
54. Owens GK, Kumar MS, and Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiological reviews 84: 767-801, 2004.
55. Ozaki T, Nishina H, Hanson MA, and Poston L. Dietary restriction in pregnant rats causes gender-related hypertension and vascular dysfunction in offspring. J Physiol 530: 141-152, 2001.
56. Rensen SS, Doevendans PA, and van Eys GJ. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Netherlands heart journal : monthly journal of the Netherlands Society of Cardiology and the Netherlands Heart Foundation 15: 100-108, 2007.
57. Roza SJ, Steegers EA, Verburg BO, Jaddoe VW, Moll HA, Hofman A, Verhulst FC, and Tiemeier H. What is spared by fetal brain-sparing? Fetal circulatory redistribution and behavioral problems in the general population. American journal of epidemiology 168: 1145-1152, 2008.
56 58. Sandoval RJ, Injeti ER, Williams JM, Georthoffer WT, and Pearce WJ. Myogenic contractility is more dependent on myofilament calcium sensitization in term fetal than adult ovine cerebral arteries. Am J Physiol Heart Circ Physiol 293: H548-556, 2007.
59. Sathishkumar K, Elkins R, Yallampalli U, and Yallampalli C. Protein restriction during pregnancy induces hypertension and impairs endothelium-dependent vascular function in adult female offspring. Journal of vascular research 46: 229-239, 2009.
60. Shadwick RE. Mechanical design in arteries. J ExpBiol 202: 3305-3313, 1999.
61. Smith JT, and Waddell BJ. Increased fetal glucocorticoid exposure delays puberty onset in postnatal life. Endocrinology 141: 2422-2428, 2000.
62. Tarry-Adkins JL, and Ozanne SE. Mechanisms of early life programming: current knowledge and future directions. Am J Clin Nutr 94: 1765S-1771S, 2011.
63. Volk KA, Roghair RD, Jung F, Scholz TD, Lamb FS, and Segar JL. Coronary endothelial function and vascular smooth muscle proliferation are programmed by early-gestation dexamethasone exposure in sheep. Am J Physiol Regul Integr Comp Physiol 298: R1607-1614, 2010.
64. Wagenseil JE, Ciliberto CH, Knutsen RH, Levy MA, Kovacs A, and Mecham RP. Reduced vessel elasticity alters cardiovascular structure and function in newborn mice. Circ Res 104: 1217-1224, 2009.
65. Wamhoff BR, Bowles DK, and Owens GK. Excitation-transcription coupling in arterial smooth muscle. Circ Res 98: 868-878, 2006.
66. Xiao D, Huang X, Pearce WJ, Longo LD, and Zhang L. Effect of cortisol on norepinephrine-mediated contractions in ovine uterine arteries. Am J Physiol Heart Circ Physiol 284: H1142-1151, 2003.
67. Yang S, and Zhang L. Glucocorticoids and vascular reactivity. Current vascular pharmacology 2: 1-12, 2004.
68. Zar JH. Biostatistical analysis. Upper Saddle River, N.J.: Prentice Hall, 1999.
69. Zeman FJ. Effects of maternal protein restriction on the kidney of the newborn young of rats. J Nutr 94: 111-116, 1968.
57 CHAPTER THREE
THE IMPACT OF PRENATAL GLUCOCORTICOID INHIBITION ON
CEREBROVASCULAR STRUCTURE AND FUNCTION IN A MODEL OF MILD
HYPOXIA-ISCHEMIA
By
Lara Durrant, Naomi Franco, Desirelys Carreon, William J. Pearce
This chapter has is unpublished as of May 2017.
58 Abstract
Neonatal stroke continues to present a major risk for infants. The present study explores the hypothesis that prenatal programming via gestational glucocorticoid inhibition results in permanent changes to cerebrovasculature structure and function during postnatal life, which fundamentally alters vascular response to a mild ischemic stroke. Pregnant female rats were administered 0.5 mg/ml Metyrapone (MET), a glucocorticoid synthesis inhibitor, from Day 11 to Day 21 of gestation. At postnatal day
9/10, pups (n=6 per group) were taken for unilateral carotid ligation. Twenty-four hours later, the pups were then exposed to 90 minutes of 8% oxygen to induce a mild hypoxic- ischemic insult. At postnatal day 11/12, middle cerebral arteries were harvested for analysis by confocal microscopy and vessel-bath myography. Mild hypoxia-ischemia significantly increased plasma corticosterone levels by 2-fold, increased passive arterial diameter by ~20%, and decreased colocalization of NM-MHC with α-actin by 4%.
Gestational MET either had no effect or reversed many of these changes, suggesting a possible protective role of glucocorticoid inhibition during gestation. These results indicate that the cerebrovasculature may be an early indicator of mild ischemic injury and may provide a potential target for future therapeutics.
Introduction
Neonatal stroke occurs at a rate of about 1 in 4000 live births (1), and the estimated mortality rate for neonates is around 3.49/100,000 annually (34). In the United
States, within the first five years of life, the cost of care per neonatal stroke patient can total more than $50,000 (18). As with adult stroke, neonatal stroke victims may suffer
59 from permanent cognitive impairment, leading to lifelong disability. Despite years of research, the care and treatment for sufferers of hypoxia-ischemia remains largely unchanged and progress has been disappointing, with clinical trials failing to show efficacy despite promise in animal models (37).
Though the underlying cause of the lack of treatment options are multifactorial and complex, the data presented in this paper will focus on three major concerns. First, animal models of hypoxia-ischemia encephalopathy (HIE) may not be as clinically relevant as hoped. Second, current models of HIE may emphasize neuronal damage at the expense of other tissues such as the cerebral blood vessels. Third, many studies may not have accounted for risk factors associated with neonatal stroke such as the possible contributions of gestational programming and early life environment. Hence, the following study presents the hypothesis that the vasculature may be one of the key mediators of neonatal hypoxia-ischemia and that programming of the long-term stress response via glucocorticoid inhibition may be instrumental in modulating this response.
One of the key challenges in neonatal stroke research is the development of an appropriate model of HIE. Prior research has demonstrated that the neonatal brain is very resistant to hypoxia. In most cases, hypoxia alone does not seem to be enough to cause ischemic injury in most cases (26). Hence, the development of specific models of HIE injury have been required. One well-characterized method of studying HIE is the Rice-
Vannucci model, which was originally developed by Levine in the 1980’s (40). The procedure involves unilateral surgical ligation of the carotid artery followed by 8% oxygen (hypoxia) for 3.5 hours. Histological characterization of the model revealed moderate to severe neuronal changes with a mortality rate of around 20%. Subsequent
60 studies have modified the HIE model, with the majority reporting rates of around 20-30% infarction (4, 5). For a variety of reasons, however, these moderate to severe infarcts may
not be representative of the actual clinical picture, as infarcts above 10% in humans are
associated with severe disability and poor clinical outcomes (17). Frequently, neonatal
stroke is not characterized by easily diagnosable symptoms such as epilepsy or sidedness.
Instead, a more accurate picture may be one where mild symptoms such as lethargy or
muscle weakness go undetected and undiagnosed until months later, prompting a
retroactive diagnosis of perinatal stroke (30, 49). As such, a model of mild hypoxia-
ischemia may be better match (33, 39, 47, 48).
Another key limitation of current neonatal stroke research is that much of the
literature is focused on neurons. This is understandable as neuronal tissue is very
metabolically sensitive and there is a well-established correlation between the severity of
neuronal cell death and subsequent neurological damage (17). The main lesion may
indeed be exacerbated by lethal levels of Ca++ and neurotransmitters (i.e. excitotoxicity),
and selective vulnerability may exist in certain sections of the brain regardless of their
perfusion status (26). However, neuroprotective strategies alone may not be enough, and
non-neuronal mechanisms may be at play. If true, then viewing stroke from a
neurovascular perspective may be helpful (3). There is evidence, for example, that
neuronal damage can be thought of as a consequence of a failed or inadequate vascular
response or impaired neurovascular response (45). The myogenic response, which serves
primarily to prevent hypo- or hyper-perfusion of downstream tissues, is an important
factor in determining what the brain ultimately “sees” during an infarct and may play a
key role in stroke (6, 15, 25). Healthy blood vessels with healthy vascular smooth muscle
61 cells that allow for appropriate contractility are critical to neuronal health and overall brain health. How a vessel responds to any given stimulus is also dependent on a multitude of factors including the different phenotypes of its corresponding smooth muscle cells (38). In the developing postnatal animal, we have previously reported that a smaller number of contractile SMCs are doing the majority of the force generation (11), such that any changes to the cell equilibrium may be detrimental to the brain’s perfusion status. Vessel growth or pruning may also play a role in overall brain health, and angiogenesis should not be ruled out (3).
Finally, data from animals from otherwise healthy or unperturbed pregnancies may not fully take into account risk factors commonly associated with clinical stroke.
While a severe stroke model may be relevant to acute birth-related trauma such as asphyxia, it may fall short of explaining the clinical picture in the presence of prenatal programming. Among the factors that influence birth outcomes and brain development, it is thought that prenatal glucocorticoid (GC) exposure plays a key role (9).
Dexamethasone and other synthetic glucocorticoids have been shown to cause a number of long-term programming changes such as high blood pressure. GC therapies have even been shown to increase adult stroke risk (14). Timing of GC administration appears to play a critical role. However, to date, most studies have focused on acute administration of glucocorticoids as opposed to chronic or sustained exposure, and fewer still have looked at the gestational effects of glucocorticoid inhibition (31). Therefore, the current study sought to investigate whether inhibition of glucocorticoid effects during gestation might produce a desirable effect postnatally and how early programming of the
62 hypothalamic pituitary adrenal (HPA) axis might affect the animal’s response to a subsequent ischemic insult.
All in all, the purpose of this study is to characterize the effects of gestational glucocorticoid inhibition on cerebrovascular structure and function within the context of a model of mild hypoxia-ischemia.
Materials and Methods
General Preparation
All experimental procedures were approved by the institutional Animal Use and
Care Committee at Loma Linda University. First-time-pregnant Sprague-Dawley rats
(Charles River Laboratories, Hollister, CA; Envigo Laboratories, Placentia, CA) were housed at constant temperature and humidity with a 12:12-h:light-dark cycle with food and water ad libidum. At day 11 of gestation until term (21 days), Metyrapone (M2696 at
0.5 mg/ml; Sigma, St. Louis, MO) was dissolved in drinking water. The water was changed out daily, and the dose was based on previous studies showing effective blockage of maternal corticosterone synthesis (42). Water consumption was monitored daily and did not differ from Control dams, which received standard filtered water. The rats were allowed to deliver spontaneously, and pups were kept with their dams until the day of surgery.
On postnatal days 9-10, two male pup litter-mates were randomly selected to undergo surgery. Male pups were chosen as sex differences have been previously demonstrated and HIE typically develops more severely in male vs. female pups (36).
One animal was anesthetized with isoflurane and underwent unilateral carotid ligation
63 using 5-0 surgical silk (Ischemia surgery), while the other underwent Sham surgery
where the carotid was dissected but not ligated. Surgical times were kept consistent
between both Sham and Ischemia animals in order to account for the potential
neuroprotective effects of isoflurane exposure (8). Following surgery, pups were allowed
to recover with their dam. At 24 hours post-surgery, the Ischemia group pups were then placed into a hypoxia chamber maintained at 8% oxygen balanced with nitrogen (heated to 37 ºC) for 90 minutes. Oxygen levels in the chamber were monitored via an oxygen meter. At the end of the hypoxia exposure, pups were allowed 15 minutes to recover under normoxic conditions (room air) before they returned to their dam. Sham animals were placed into a separate temperature-controller chamber under normoxic conditions for the same duration.
The overall study design included four groups of Sprague-Dawley rats: 1) Sham- operated animals (Sham), 2) Ischemia animals that underwent carotid ligation surgery followed by hypoxia (Ischemia), 3) Sham animals that were administered Metyrapone during gestation (Sham+MET), and 4) Ischemia animals that received Metyrapone during gestation (Ischemia+MET). At postnatal days 11-12, all pups were weighed, anesthetized with 100% CO2 (1 minute) and then decapitated. The brains were rapidly removed and placed in ice-cold physiological salt solution (PSS) containing (in mM): 130 NaCl, 10.0
HEPES, 6.0 glucose, 4.0 KCl, 4.0 NaHCO3, 1.8 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, and
0.025 EDTA, at pH 7.4. The main branch of the middle cerebral artery (MCA) was taken beginning at its origin next to the Circle of Willis, cleaned of all connected tissue, cut into length of 3-5 mm, and then mounted on glass cannulas on a CH-1 vessel-chamber positioned on the stage of an inverted microscope (Living Systems, Burlington, VT). The
64 proximal cannula was connected to a pressure transducer and a windkessel reservoir of
PSS, used to precisely set and change the transmural pressures of the arteries. The distal
cannula was connected to a Luer-lock valve that was opened to gently flush the lumen
during the initial equilibrium. Vessel viability was confirmed by the presence of
contraction to high K+ solution. After subsequent equilibration, the outflow valve was
closed, and all measurements were conducted under stop-flow conditions. Arterial
diameters were recorded with the SoftEdge Acquisition Subsystem (IonOptix, Milton,
MA). All methods used in this study have been previously described in detail (11, 12).
Measurement of Smooth Muscle Calcium Concentration
Once cannulated and equilibrated at 21 ºC, the artery segments were loaded with
Fura-2 AM (ThermoFisher Scientific, Waltham, MA) for 20 minutes at a concentration of
1 uM, as previously described (11, 12). After loading, the arteries were washed with PSS,
and the bath temperature was increased to 37 ºC. Photons emitted at 510 nm were
detected at a sample rate of 3 Hz using an IonOptix photomultiplier system that
automatically corrects for background fluorescence. Kd values obtained from separate in
2+ vitro calibrations were used to convert the experimental fluorescent ratios (R) to [Ca ]i
2+ over the physiological range by iterative fit to the Grynkiewicz equation: [Ca ]i = Kd[(R-
Rmin)/(Rmax-R)]Sf (20). Our calculations using this equation employed the following
averaged values: Sf (5635), Rmi n (0.468), Rma x (6.837), Kd (251 mM).
65 Fluorescent Immunohistochemistry
Arteries were fixed in 4% neutral buffered EM-grade formaldehyde (Electron
Microscopy Sciences, 15713-S) overnight, and then dehydrated, embedded in paraffin, and cut into sections 5 µm thick. The sections were then deparaffinized, rehydrated, immersed in citrate buffer at 6.03, and microwaved for 5 minutes to facilitate antigen recovery. The sections were then incubated in 1% BSA (SC-2323; Santa Cruz
Biotechnology, Santa Cruz, CA), with Triton X-100 (T-8787; Sigma-Aldrich) blocking buffer for hour and a half. Next, the sections were double-stained with primary antibodies reactive with smooth muscle ɑ-actin (A5228 @ 1:600; Sigma-Aldrich), smooth muscle 2
(SM2) myosin (Ab53219 @ 1:400; Abcam, Cambridge, MA), and SMemb myosin
(Poly19099 @ 1:300; BioLegend, San Diego, CA), applied and incubated at 4 ºC overnight. The following day, the sections were incubated for two hours at room temperature with two secondary antibodies labeled with Dylight-488 and Dylight-633, and then examined with our Olympus FV1000 confocal microscope at an optical section thickness of 1 µm, a lateral resolution between 146 and 185 nm, an axial resolution between 545 and 693 nm, and a numerical aperture of 1.4.
Confocal images of coronal artery sections were analyzed using Image-Pro (v5.9;
Media Cybernetics, Silver Spring, MD) and the degree of colocalization was calculated using data obtained from CoLocalizer Pro (Version 2.6.1, CoLocalization Research
Software). For the colocalization analysis, coronal sections were extracted from the parent images via Adobe Photoshop (Version CS6; Adobe Systems, San Jose) using a masking routine to eliminate background contributions to the measurement statistics.
These methods have previously been described in detail (11, 12).
66 ELISA
Whole trunk blood was collected at the time of sacrifice into MiniCollect Lithium
Heparin blood collection tubes (Greiner Bio-One, Kremsmunster, Austria). Blood was
collected between the hours of 10 AM-12 PM; the times were kept consistent in order to account for any variability due to circadian rise and fall of corticosterone levels. The blood was then centrifuged at 2000 RPM at 4 ºC for 15 minutes to separate the plasma, and then aliquoted out and frozen at -20 ºC. In order to release any corticosterone bound by protein, each plasma sample was treated with perchloric acid (HClO4) followed by
KOH, and an ethyl acetate extraction was performed using a speed vacuum (SPD 121P-
115). Corticosterone levels were then measured with an ELISA kit (ADI-900-097, Enzo
Life Sciences). Samples were read using a Gen5 1.11 BioTek microplate reader.
Chemicals and Reagents
All of the chemicals and reagents used in this study were from Sigma-Aldrich (St.
Louis, Missouri) or Thermo Fischer Scientific (Waltham, Massachusetts), unless
otherwise noted.
Data Analysis and Statistics
All values are given in the text as means ± SE, and statistical significance implies
P < 0.05 unless stated otherwise. In all cases, n refers to the number of animals studied.
Values of artery diameter, and wall calcium were analyzed via two-way ANOVA with
repeated measures using treatment (Sham vs Ischemia) or Metyrapone status (+/- MET)
and pressure as factors. Post hoc comparisons were performed using the Fisher Paired
67 Least Significant Difference analysis. Values for the Colocalization Coefficient were
compared using a Behren’s-Fischer analysis with pooled variance. For analysis of
myosin isoform expression, all image pixels with greater than median intensity for α- actin were segregated into two groups with intensities either above or below the median intensity for each myosin isoform, respectively (SM-MHC, SMemb). The percentages of total pixels in each of these groups was calculated for each age group, and then compared between age groups using a Behren’s-Fischer analysis with pooled variance. All data sets were normally distributed, as verified using a D’Agostino-Pearson K2 test, and homogeneity of variance within ANOVA was verified using a Bartlett’s-Cochran test
(50).
Results
This study is based on data obtained from litters of rat pups from four distinct treatment groups; Sham and Ischemia animals whose mothers received normal drinking water, as well as Sham+Metyrapone and Ischemia+Metyrapone animals whose mothers received 0.5 mg/ml of Metyrapone during days 11 through 21 of gestation. Left and right artery segments from each animal (n=6 per group) were used for contractility studies, and two adjacent artery segments were taken for immunohistochemistry and confocal imaging. A separate set of animals (n=5 per group) were taken for in vivo MRI, transcardially perfused. The brains were then removed, cut into sections, and then stained for a number of markers including FluoroJade-C, c-fos, IBA-1, and GFAP.
68 Plasma Corticosterone in a Model of Mild Hypoxia Ischemia
Plasma corticosterone levels were measured from samples of whole trunk blood collected at age P11/P12 upon sacrifice 24 hours post-hypoxia (48 hours post-surgery).
Samples were extracted using an ethyl acetate protocol and processed with an ELISA kit.
Results were calculated based on a standard curve of varying levels of corticosterone that
was run concurrently with the samples. All samples were run in duplicate. Corticosterone
levels from Ischemia animals (3000 ± 1416 pg/ml) were significantly greater than Sham
(1553 ± 650 pg/ml) animals by ~2-fold. Samples from Ischemic animals gestationally
treated with Metyrapone (Ischemia+MET) (1638 ± 1093 pg/ml) were not significantly
different than levels from Sham+Metyrapone animals (1257 ± 108 pg/ml). This data was
collected from n=4 animals per group.
69
Figure 1. Plasma Corticosterone Levels in a Model of Mild Ischemia. Presented above is data from n=4 animals per group. In Figure 1, plasma corticosterone levels were measured from samples collected from pups at various time points. All samples were extracted using an ethyl acetate protocol and processed with an ELISA kit. Figure 1A shows a time course showing plasma corticosterone levels 0, 2, 26, and 48 hours post-surgery (Note: 0-hours post-surgery animals were treatment-naïve). Two hours post-surgery, Ischemic animals showed increased plasma corticosterone levels compared with Ischemic animals whose dams were exposed to MET during gestation. At 26 hours, this trend was reversed. It was found that plasma corticosterone levels of Ischemic animals were significantly lower than in Sham animals. There were no significant differences between Sham vs. Sham+Metyrapone. Figure 1B shows plasma corticosterone levels from all four treatment groups at the 48 hour mark. Ischemia plasma corticosterone levels were increased ~2-fold, compared with Sham. There were no differences between Sham and Sham+MET groups.
70 Effects of Changes in Pressure on Maximum Passive Diameter and Compliance in
Sham, Ischemia, Sham+Metyrapone and Ischemia+Metyrapone Pup MCAs
In Sham arteries exposed to EGTA, there was an increase in maximum passive diameter from 165 ± 5 µm at 20 mm Hg to 186 ± 4 µm at 80 mmHg (Figure 2, upper left panel). There was also an upward shift in the maximum passive diameter in Ischemia arteries, ranging from a diameter of 194 ± 8 µm at 20 mm Hg and 207 ± 7 µm at 80 mm
Hg. Ischemia artery passive diameters were significantly higher than Sham arteries at 20 mm Hg, 40 mm Hg, and 60 mm Hg of pressure. With respect to Metyrapone treated arteries, we saw an increase of passive diameters from 179 ± 6 µm at 20 mm Hg to 190 ±
6 µm at 80 mm Hg (Figure 2, upper right panel). Ischemia+MET arteries showed a slight upward shift compared to Sham+Metyrapone arteries, starting at 189 ± 6 µm at 20 mm
Hg and reaching a maximum of 203 ± 7 µm at 80 mm Hg. This effect only reached statistical significance at 80 mm Hg. Sham and Sham+Metyrapone arteries differed significantly at low pressures (20, 40, and 60 mm Hg), with Sham+Metyrapone arteries trending to high maximum passive diameters.
Average values of arterial compliance (Figure 2, lower panels), calculated as the derivative of the relation between passive diameter and pressure (Figure 2, upper panels), ranged from 0.18 % diameter / mm Hg at 20-40 mm Hg to 0.07 % diameter / mm Hg at
60-80 mm Hg. All groups showed a downward sloping pattern with the least compliance
(% diameter / mm Hg) at 60-80 mm Hg. With respect to Sham vs Ischemia arteries, there were significant differences at 40-60 mm Hg with Sham arteries having a compliance of
0.080 ± 0.013 % diameter / mm Hg vs. Ischemia with 0.051 ± 0.009 % diameter / mm
Hg, as well as at 60-80 mm Hg with 0.79 ± 0.022 % diameter / mm Hg for Sham and
71 0.042 ± 0.008 % diameter / mm Hg for Ischemia. With respect to Sham+Metyrapone vs.
Ischemia+Metyrapone arteries, we saw no significant differences at 20-40 and 40-60 %
diameter / mm Hg, but we did see a significant difference at 60-80 mm Hg with
Sham+Metyrapone arteries showing an average compliance of 0.042 ± 0.008 % diameter
/ mm Hg and Ischemia + Metyrapone arteries showing a compliance of 0.064 ± 0.008 %
diameter / mm Hg. Sham+MET arteries were significantly less compliant compared with
Sham arteries at 60-80 mm Hg, with Sham arteries showing a compliance of 0.079 ±
0.013 % / mm Hg vs 0.073 ± 0.009 % / mmHg for Sham+MET arteries. Overall,
Ischemia appeared to raise maximum passive diameters and to decrease compliance.
Treatment with Metyrapone increased passive diameters at low pressures and compliance
at high pressure. When MET animals were exposed to Ischemia, MET did not exacerbate the increase in diameters seen in Control Ischemic arteries.
72
Figure 2. Passive Diameter and Incremental Compliance in Ischemia and MET arteries. After incubation in 3 mM EGTA solution to eliminate all active tone, passive diameters increased with intraluminal pressure in all groups (Upper Panels). At low pressures (20 mm Hg, 40 mm Hg, and 60 mm Hg), diameters in Ischemia arteries were greater than in Sham arteries (Upper Left Panel). By contrast, Sham+MET and Ischemia+MET arteries did not differ significantly except at 80 mm Hg, indicating that Metyrapone eliminated most of the differences between Sham and Ischemia. Sham vs Sham+MET arteries differed at low pressures (20 mm Hg, 40 mm Hg, 60 mm Hg), indicating a treatment effect of MET (Upper Right Panel). When the slopes of the normalized diameter to pressure (incremental compliance) were calculated, compliance decreased for all arteries with increasing pressure (Lower Panels). Between 40-60 mm Hg and 60-80 mm Hg pressure, Ischemia arteries were significantly less compliant than Sham arteries (Lower Left Panel). In animals treated with 0.5 mg/ ml MET during gestation, this trend was eliminated between 40-60 mm Hg pressure and reversed between 60-80 mm Hg, with Ischemia+Metyrapone arteries showing greater incremental compliance than Sham+Metyrapone arteries (Lower Right Panel). Sham and Sham+MET arteries differed at 60-80 mm Hg, indicating a treatment effect at high pressures. Error bars indicate SEM for n values of 6 in all groups. Asterisks (*) indicate significant differences (p < 0.05) between Sham and ICH groups at corresponding pressures, while daggers (†) indicate differences between Sham and Sham+Metyrapone groups.
73 Changes in Diameter, Calcium, and Calcium Sensitivity Induced by Pressure and K+ in
Pup MCAs
Pressure-induced changes in diameter (ΔD) were calculated as the difference in transmural diameter in arteries in 3 mM EGTA solution (passive tone) minus the diameter in PSS (active tone). Plots of these values vs pressure were used to calculate the area under the curve. The results of these calculations are shown in Figure 3 (upper left panel). No significant differences were observed between any of the four groups with regards to pressure-induced contractions. As before with PSS-induced contractions, potassium-induced ΔD was measured as the difference in diameter in PSS + 120 mM
KCl vs in 3 mM EGTA solution. Plots of these values versus intraluminal pressure were used to calculate the area under the curve. As shown in Figure 3 (upper right panel), the only significant differences seen were between Control vs Ischemia+MET group arteries, with Ischemia+MET arteries showing significantly depressed potassium-induced
contractions compared with control Ischemia arteries by about 37% indicating an effect
of the gestational MET-treatment on Potassium-induced contractility.
Pressure-induced changes in wall calcium were calculated as the percent
difference between wall calcium in PSS and wall calcium in 3 mM EGTA solution. Plots
of these values versus intraluminal pressure (Δ Wall Calcium-Pressure) were used to
calculate the area under the Δ Calcium curve, the results of which are shown in Figure 3
(middle left panel). In Ischemia arteries, we saw a significant increase in pressure-
induced changes in wall calcium by 8.2% (middle left panel). This effect was absent in
animals treated with Metyrapone during gestation. As with pressure-induced changes in
wall calcium, potassium-induced changes in wall calcium were calculated as the percent
74 difference in wall calcium of arteries in PSS + 120 mM KCl vs. 3 mM EGTA solution.
As before, the plot of these values against pressure (Δ Wall Calcium-Pressure) was used
to calculate the area under the curve for potassium-induced contractions. As shown in
Figure 3 (middle right panel), Ischemia significantly increased potassium-induced
calcium contraction by 11.5% compared with Sham arteries. Arteries treated with
Metyrapone from both the Sham and Ischemia groups showed no significant differences,
indicating that the differences in potassium-induced contractions seen in Sham vs
Ischemia arteries was absent following Metyrapone treatment during gestation.
The contractile force generated by smooth muscle can be calculated from the
product of cytosolic calcium concentration and myofilament calcium sensitivity
(Contractile Force = Ca++ x Ca++ Sensitivity). By taking the ratio of Δ Diameter to Δ Wall
Calcium, as calculated individually at each pressure step, a step-wise estimate of the
calcium sensitivity can be determined. Plots of the Calcium Sensitivity (ΔD/ΔWall
Calcium-Pressure) were used to calculate the area under the curve. The results of these
calculations are shown in Figure 3 (bottom left panel). We did not find any significant
differences between either Sham vs Ischemia or in arteries from Metyrapone-treated
pups, though there was a significant difference between Sham vs Sham+MET arteries,
with Sham+MET showing a depression of calcium sensitivity by 34.7%. Similarly, the
plot of ΔD/ΔWall Calcium vs. Pressure for K+-induced contractions was used to generate the area under the curve for each pressure step. As before, we did not see any significant
differences between any groups, Sham or Ischemia, with or without Metyrapone. We also
did not see any significant differences between Sham vs Sham+MET, though there was a
75 significant decrease in Potassium-induced calcium sensitivity between Ischemia vs
Ischemia+MET groups, likely indicating programming effects by Metyrapone.
76
Figure 3. Changes in Diameter, Calcium, and Calcium Sensitivity Induced by Pressure and K+ in Ischemia and MET arteries. Pup middle cerebral artery contractility was assessed by measuring changes in response to increasing intraluminal pressures (left panels) an in PSS with 120 mM K+ (right panels). Three main parameters were measured, including transmural pressure (Δ Diameter), wall calcium concentration (Δ Wall Calcium), and calcium sensitivity (Δ Diameter / Δ Wall Calcium), the ratio of diameter to wall calcium. All responses were measured vs intraluminal pressure and the area under the curve (AUC) was calculated for statistical comparison purposes. Here we show the area under the curve for Δ Diameter vs Pressure (Upper Panels), Δ Wall Calcium vs Pressure (Middle Panels), and Δ Diameter / Δ Wall Calcium (Lower Panels). Neither untreated nor Metyrapone- treated arteries showed any significant differences in the Δ Diameter responses (Upper Panels). Ischemia significantly increased Δ Wall Calcium when compared with Sham arteries, while this trend was reversed in arteries exposed to K+-induced contractions (Middle Panels). With respect to Metyrapone, we saw no significant differences in either Pressure-Induced or K+-induced contractions, though like Ischemia arteries, there was a significant increase compared to Sham arteries with Pressure, and a decrease during Potassium-induced contraction. There were no significant Sham vs Ischemia differences in the responses of myofilament calcium sensitivity in either Control or MET arteries. Asterisks (*) indicate significant differences (p < 0.05) between Sham and ICH groups at corresponding pressures, while daggers (†, ‡) indicate differences between Sham and Sham+Metyrapone groups, and Ischemia vs Ischemia+Metyrapone, respectively.
77 Effects of Ischemia ± Metyrapone on Markers of Smooth Muscle Cell Differentiation
in Pup MCAs
To obtain a more complete assessment of the effects of gestational Metyrapone and Ischemia on contractile function, contractile protein organization was examined via confocal microscopy of two different marker pairs: a) Non-Muscle Myosin Heavy Chain
(NM-MHC, “SM-Embyronic”) and Smooth Muscle α-Actin (αActin) and b) Smooth
Muscle Myosin Heavy Chain (SM-MHC) and αActin (Figure 4). For each protein pair examined, only pixels for which both proteins were above a set threshold intensity were examined, and the total number of pixels was defined as 100%. The pixels were then put into three different categories: 1) Low MHC / Low αA (Low/Low), where both MHC and
αA were above our minimum threshold but below our set threshold, 2) Low MHC / High
αA (Low/High), where MHC was above minimum threshold and αA was above the set maximum threshold, and 3) High MHC / High αA (High/High), where both MHC and αA were above set threshold. The effects of Ischemia and gestational Metyrapone on contractile protein organization were examined by looking at the distribution of pixels across each category.
78
Figure 4. Effects of Ischemia ± Metyrapone on Markers of Smooth Muscle Cell Differentiation. The effects of Ischemia with and without Metyrapone were examined in order to determine the colocalization of smooth muscle cell differentiation markers. Arteries were embedded in paraffin and cut into 5 µm thick sections. Colocalization was determined by confocal microscopy for two marker pairs: a) Non-Muscle Myosin Heavy Chain (NM-MHC) and Smooth Muscle a-Actin (αActin) and b) Smooth Muscle Myosin Heavy Chain (SM-MHC) and αActin. Shown above are representative sections from each of the four treatment groups (n = 6 animals). For all colocalization done, α-Actin was placed in the 488 nm (red) channel because it was the most abundant protein, while the least abundant protein was placed into the 633 nm (green) channel.
79 Effects of Ischemia ± Metyrapone on Colocalization of MHC Isoforms with Smooth
Muscle α-Actin
In sections stained for both SM-MHC and αActin (Figure 5, Upper Panels), there were no significant differences in any of the categories (Low/Low, Low/High, or
High/High) when comparing Control Sham vs Ischemia arteries. By comparison in the
Metyrapone-treated groups, there was a upward shift of pixels in response to Ischemia, with the Low/Low category showing an average increase of 6.7% by Ischemia and a subsequent decrease of 5.9% in the Low/High category. There were no changes in the
High/High category. MET arteries showed a significant rightward shift compared with
Sham arteries, with a significant decrease by 8.3% in the Low / Low category, as well as an increase in the Low/High and High/High categories by 6.3 and 2.0 %, respectively. As for arteries double stained with NM-MHC and αActin (Figure 5, Lower Panels), there were significant changes between Sham and Ischemia in Low/Low and High/High categories, with Ischemia arteries generally showing a leftward shift towards Low/Low categories: Ischemia arteries in the Low/Low category showed a significant increase by
7.8% and a significant decrease of 4.0 % in the High/High category compared with Sham arteries. Metyrapone showed a different trend, with significant increases in the Low-Low category and decreases in the Low/High category; unlike with Control arteries, there were no significant differences between Sham+MET vs Ischemia+MET in the High/High category. With respect to MET-treatment alone, Sham vs Sham+MET arteries showed no differences except in the High/High category, with Sham+MET arteries showing a decrease of 2.7% compared with Sham. Compared with Ischemia arteries,
Ischemia+MET arteries also showed a small, albeit significant increase in the High/High
80 category by 1.2%. Ischemia alone seems to support differentiation into a de-differentiated phenotype, while gestational treatment with Metyrapone seemed to reverse this effect, encouraging a more contractile phenotype.
81
Figure 5. Effects of Ischemia ± Metyrapone on Colocalization of MHC Isoforms with Smooth Muscle α-Actin. The figure above shows the pixel distributions for our four experimental groups with two different marker pairs: a) Smooth Muscle Myosin Heavy Chain (SM-MHC) and Smooth Muscle a-Actin (α-Actin) and b) Non-Muscle Myosin Heavy Chain (NM-MHC) with α-Actin. Within each marker pair, the total of all pixels across each of the three distributions (Low MHC/ Low αA, Low MHC/ High αA, High MHC, High αA) is 100%. Please note that the vertical scale for each of the three distributions are different (100% vs. 50% vs. 20%). Ischemia had no effect on pixel distributions with arteries stained for SM-MHC, but caused a leftward shift towards High MHC / High aA to Low MHC / Low αA in the NM-MHC category. Treatment with Metyrapone during gestation generally showed a reversed trend, with pixel distributions moving away from the Low MHC / High αA category during Ischemia toward Low MHC / Low αA. In the NM-MHC category, there was also an increase in pixel distributions to High MHC/ High αA. Error bars indicate SEM with n=6 animals in all groups. Asterisks (*) indicate significant differences (P<0.05) between Sham and Ischemia groups, while daggers (†, ‡) indicate differences between Sham and Sham+Metyrapone, and Ischemia and Ischemia+Metyrapone groups, respectively.
82 Discussion
The present study explores the hypothesis that glucocorticoid inhibition during gestation can influence cerebrovascular structure and function relations during postnatal life, and that this altered glucocorticoid profile can modify the vascular response to mild hypoxia-ischemia. Despite the use of a model of mild hypoxia-ischemia, there were key changes to vascular structure and function, indicating that vascular behavior may be different even in the absence of overt histological changes. Metyrapone, when administered during gestation, acted to counteract many of these effects. Together, these results suggest that a model of mild hypoxia-ischemia may be an additional method by which to study stroke in the neonate, and that glucocorticoid programming plays a key role in the ability of the cerebrovasculature to respond to a stressful stimulus.
In this model of mild hypoxia-ischemia, corticosterone levels at 2 hours post- surgery were significantly elevated compared with MET-treated post-surgery animals.
Additionally, at the 24 hour post-hypoxia time period (48 hr post-surgery), there was a significant ~2-fold increase corticosterone in Ischemia arteries compared with Sham,
Sham+MET, and Ischemia+MET groups (Figure 1). In the literature, there is considerable variability with respect to neonatal plasma corticosterone levels, with resting values ranging from as low as 1 ng/ml to about 60 ng/ml. In rodents, this might be partially explained by the hyporesponsive period (SHRP). As the name implies, this is a period during the early postnatal life of rodents in which time the HPA axis system is hyporesponsive to stressors. Basal corticosterone levels may become depressed and stay this way until around two weeks of age (32), though stressors can still elicit a response.
Corticosterone levels that increased by about 2-fold are not unexpected and may indicate
83 that, while mild, hypoxia at 24-hours post-ischemia is a stress to the pups (22). Likewise,
the finding that gestationally-treated MET animals also showed decreased corticosterone
levels at the 2 and 48 hour time periods suggest that HPA axis reactivity was significantly
altered by gestational inhibition of GCs. This is in agreement with results from other
studies where acute administration of MET was found to decrease corticosterone levels in
rat adults (21, 29) and pups (10, 28). In a pig model, it was found that administration of
Metyrapone during gestation lowered plasma cortisol levels at postnatal day 14 (44).
These findings are also in line with studies that looked at chronic glucocorticoid exposure
during gestation, and found altered HPA axis reactivity into adulthood (7).
Rat middle cerebral arteries taken for analysis by vessel-bath myography from the four different treatment groups revealed structural differences under pressure-passive conditions (Figure 2). Arteries from the Ischemia group were found to have increased passive diameters as well as decreased compliance and this phenotype was partially rescued by MET-treatment during gestation. The addition of gestational MET appears to have had an inhibitory effect on the normal ischemia-induced increase in stiffness. This
could indicate that programming effects have occurred well before the hypoxic-ischemic
insult could play a role. In the literature, this is a common finding with respect to excess
antenatal glucocorticoid treatments, with evidence that high glucocorticoid exposure can
induce vascular aging and stiffening effects (16, 27), perhaps through changes in vessel
collagen, elastin, or extracellular matrix protein levels (13). The reverse experiment,
however, with the inhibition of endogenous glucocorticoid has not been studied in any
great detail, and the present study will be one of the first to publish such findings with
respect to the neonatal cerebrovasculature. As for the mechanisms, it is likely that acute
84 stress caused by the hypoxic-ischemic insult contributed to the ischemia-induced increase in vascular stiffness. MET-treated animals, by contrast, may not have been exposed to the same rise in glucocorticoid levels, as shown by the time-course in Figure 1. This suggests that arterial stiffening may have been prevented due to MET-induced changes in
HPA reactivity.
In addition to measuring vessel behavior under passive pressure conditions, vessel myography is a technique that can also be used to investigate active contractility when the vessel is exposed to various external conditions (Figure 3). By the use of the intracellular fluorescent Ca++ dye fura-2, vessel contractility may also be recorded concurrently with measures of intracellular wall calcium. Together, these measurements provide insight into the functional behavior of our arteries. Despite significant differences in total wall calcium levels in pressure-induced contractions when comparing
Sham and Ischemia arteries, these results did not translate to any overall differences in contractility or calcium sensitivity.
This is in line with other studies, where intracellular calcium levels post-hypoxia were found to be increased in neuronal somata and axons during the first 24-hours of recovery (19). As for why there were no net changes in contractility between Sham and
Ischemia arteries, it is possible that the hypoxia-ischemia model used in this study may simply be too mild or that more statistical power (i.e. a larger cohort) is needed to detect a possible upward trend of pressure-induced diameter changes. It is also possible that other factors such as post-injury compensatory mechanisms may be at play or that the timing of the injury at 24-hours post-hypoxia may play a role. With respect to
Metyrapone-treated Sham arteries compared with Sham arteries, there were significant
85 increases to vessel wall calcium without a significant loss or gain in contractility, indicating that calcium sensitivity may have been lost due to the programming effects of
MET. This is in line with other work where it was found that corticosterone and dexamethasone both were shown to reduce intracellular calcium concentrations in astrocytes and neurons (43), suggesting that glucocorticoids may have an inhibitory effect on calcium sequestration or extrusion on cerebral smooth muscle cells as well. While the plasma corticosterone levels in Gestational MET-treated animals was not significantly less at the 24-hour post-hypoxia time point, the time-course presented in this study
(Figure 1) indicates that the total exposure to circulating corticosterone levels post-injury was less than in control Ischemia animals. Hence, the normal inhibitory effect of corticosterone may have been temporarily lifted due to the programming-induced HPA axis changes, leading to an increase in intracellular levels of calcium in MET arteries.
Whereas myogenic tone can be used as a measure of active vessel tone, it can also be helpful to look at maximum tone, such as from exposure to a high K+ solution. High levels of potassium act in a receptor-independent manner and stimulate vascular smooth muscle cells towards a maximally contractile state, also known as maximum tone. In the groups studied, the only significant differences were seen with respect to Ischemia and
Ischemia+MET treatments, with Ischemia+MET arteries showing a significant depression in both potassium-induced contractility and calcium sensitivity vs in control
Ischemia arteries. This occurred without any significant changes to wall calcium, which likely contributed to the significant loss of potassium-induced calcium sensitivity in
Ischemia+MET arteries, at least compared with control Ischemia arteries. This indicates that the vessel responses to high potassium conditions has been fundamentally altered by
86 programming effects. Little work has been done with the effects of glucocorticoid inhibition on vessel contractility. One lab found that antenatal programming of glucocorticoids has previously been shown to alter coronary artery contractility to acetyl choline in lambs, and it did not affect KCl-induced contractions (41). However, our study did not document any significant differences in potassium-induced contractility with the deprivation of glucocorticoids during gestation which may be related to the difference in experimental design (inhibition vs administration of glucocorticoids).
Changes in arterial structure and function co-occur with corresponding changes in vascular smooth muscle cell phenotype (Figures 4 & 5). Likewise, transitions of smooth muscle from one phenotype to another can in turn influence arterial structure and function as well as calcium handling (23, 35, 46). Such changes in differentiation patterns can be identified by looking at contractile protein expression (2, 11, 24). The two markers chosen for this study were Smooth Muscle Myosin Heavy Chain (SM-MHC, “SM2”) and
Non-Muscle Myosin Heavy Chain (NM-MHC), both colozalized with and double-stained with α-actin. SM2 is a marker for fully differentiated contractile smooth muscle, while
NM-MHC is considered a marker of developing or immature vascular smooth muscle.
Together, they form a window into the vascular responses to hypoxic-injury and the programming induced by administration of Metyrapone during gestation. With respect to
SM-MHC, we see no significant differences between Control Sham and Ischemia arteries, indicating that Ischemia alone does not seem to be contributing to differentiation of the smooth muscle into a more contractile phenotype. It is possible that changes may occur over time throughout the life of the animal; an aging study might be beneficial to look at the long-term effects of mild ischemia on cerebrovascular health. Nevertheless,
87 there is a significant effect of Metyrapone, which appears to be causing a shift to a more contractile phenotype, which then appears to be reversed when the arteries are taken from
Ischemia+MET animals. From this data, we expected that Sham+MET arteries might show increased contractility vs Sham arteries. However, we did not see this behavior.
Instead, what we see is that Sham+MET arteries appear to have increased pressure- induced vessel wall calcium and decreased calcium sensitivity, while base contractility remained unchanged. Finally, Ischemia in combination with gestational glucocorticoid inhibition appears to have had a normalizing effect on smooth muscle cell differentiation, contributing to a new profile altogheter.
With respect to NM-MHC (SM-Embryonic), we see a slightly different pattern. In this case, there is a leftward shift of pixels in response to Ischemia, perhaps indicating a shift to a less contractile, less differentiated phenotype. A similar pattern can be seen with arteries from gestational Metyrapone-treated pups. In response to Ischemia, there was a shift leftward and to the middle, suggesting a partially-differentiated or transition state, most likely in response to the hypoxic-ischemic injury. Again, Metyrapone administration appeared to have a normalizing, or perhaps stabilizing effect on the vascular response to Ischemic stress. From the perspective of the artery, MET treatment is protective against further damage via Ischemia, at least in the High NM-MHC/High
NM-MHC category. Together, this data indicates that gestational Metyrapone, and likewise antenatal glucocorticoid status, may play a role in the development of normal artery physiology and the vascular response to injury.
88 Conclusions
In conclusion, mild hypoxia-ischemia is a viable model by which to study vascular changes, and vice versa. The data presented in this paper provides support for the idea that vascular changes can occur even under very mild conditions, and that early
life (mid-late gestational) glucocorticoid status can modify vascular responses even
during postnatal life. There are several limitations to this study. Future work will be
needed to determine precisely how programming by corticosterone is acting to alter
structure-function relations in the cerebrovasculature and, subsequently, alter the pup’s response to hypoxia-ischemia. Special attention will need to be placed on exploring
possible epigenetic mechanisms including miRNA activity. Likewise, the precise nature
of the cerebrovascular changes merit further study, potentially with the help of organ
culture work, investigating other markers of contractility, or perhaps delving deeper into
calcium handling mechanisms like calcium sensitivity, influx, or release.
Acknowledgments
Special thanks to the laboratories of Dr. Obenaus and Dr. Buchholz.
89 References
1. Aden U. Neonatal stroke is not a harmless condition. Stroke; a journal of cerebral circulation 40: 1948-1949, 2009.
2. Adeoye OO, Butler SM, Hubbell MC, Semotiuk A, Williams JM, and Pearce WJ. Contribution of increased VEGF receptors to hypoxic changes in fetal ovine carotid artery contractile proteins. Am J Physiol Cell Physiol 304: C656-665, 2013.
3. Arai K, Jin G, Navaratna D, and Lo EH. Brain angiogenesis in developmental and pathological processes: neurovascular injury and angiogenic recovery after stroke. The FEBS journal 276: 4644-4652, 2009.
4. Ashwal S, Ghosh N, Turenius CI, Dulcich M, Denham CM, Tone B, Hartman R, Snyder EY, and Obenaus A. Reparative effects of neural stem cells in neonatal rats with hypoxic-ischemic injury are not influenced by host sex. Pediatr Res 75: 603-611, 2014.
5. Ashwal S, Tone B, Tian HR, Chong S, and Obenaus A. Comparison of two neonatal ischemic injury models using magnetic resonance imaging. Pediatr Res 61: 9-14, 2007.
6. Baek EB, and Kim SJ. Mechanisms of myogenic response: Ca(2+)-dependent and - independent signaling. Journal of smooth muscle research = Nihon Heikatsukin Gakkai kikanshi 47: 55-65, 2011.
7. Bertram CE, and Hanson MA. Prenatal programming of postnatal endocrine responses by glucocorticoids. Reproduction 124: 459-467, 2002.
8. Burchell SR, Dixon BJ, Tang J, and Zhang JH. Isoflurane provides neuroprotection in neonatal hypoxic ischemic brain injury. J Investig Med 61: 1078- 1083, 2013.
9. Chang YP. Evidence for adverse effect of perinatal glucocorticoid use on the developing brain. Korean journal of pediatrics 57: 101-109, 2014.
10. Charles MS, Ostrowski RP, Manaenko A, Duris K, Zhang JH, and Tang J. Role of the pituitary-adrenal axis in granulocyte-colony stimulating factor-induced neuroprotection against hypoxia-ischemia in neonatal rats. Neurobiology of disease 47: 29-37, 2012.
11. Charles SM, Zhang L, Cipolla MJ, Buchholz JN, and Pearce WJ. Roles of cytosolic Ca2+ concentration and myofilament Ca2+ sensitization in age-dependent cerebrovascular myogenic tone. Am J Physiol Heart Circ Physiol 299: H1034-1044, 2010.
12. Charles SM, Zhang L, Longo LD, Buchholz JN, and Pearce WJ. Postnatal maturation attenuates pressure-evoked myogenic tone and stretch-induced increases
90 in Ca2+ in rat cerebral arteries. Am J Physiol Regul Integr Comp Physiol 293: R737- 744, 2007.
13. Chuang TD, Pearce WJ, and Khorram O. miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids. Am J Physiol Cell Physiol 309: C117-125, 2015.
14. Craft TK, and Devries AC. Vulnerability to stroke: implications of perinatal programming of the hypothalamic-pituitary-adrenal axis. Frontiers in behavioral neuroscience 3: 54, 2009.
15. Davis MJ, and Hill MA. Signaling mechanisms underlying the vascular myogenic response. Physiological reviews 79: 387-423, 1999.
16. Durrant LM, Khorram O, Buchholz JN, and Pearce WJ. Maternal food restriction modulates cerebrovascular structure and contractility in adult rat offspring: effects of metyrapone. Am J Physiol Regul Integr Comp Physiol 306: R401-410, 2014.
17. Filippi CG, El-Ali AM, Miloushev VZ, Chow DS, Guo X, and Zhao B. Computer-assisted volumetric measurement of core infarct volume in pediatric patients: feasibility for clinical use and development of quantitative metrics for outcome prediction. AJNR Am J Neuroradiol 36: 789-794, 2015.
18. Gardner MA, Hills NK, Sidney S, Johnston SC, and Fullerton HJ. The 5-year direct medical cost of neonatal and childhood stroke in a population-based cohort. Neurology 74: 372-378, 2010.
19. Grow J, and Barks JD. Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts. Clinics in perinatology 29: 585-602, v, 2002.
20. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. The Journal of biological chemistry 260: 3440-3450, 1985.
21. Habib S, Gattineni J, Twombley K, and Baum M. Evidence that prenatal programming of hypertension by dietary protein deprivation is mediated by fetal glucocorticoid exposure. American journal of hypertension 24: 96-101, 2011.
22. Hairston IS, Ruby NF, Brooke S, Peyron C, Denning DP, Heller HC, and Sapolsky RM. Sleep deprivation elevates plasma corticosterone levels in neonatal rats. Neurosci Lett 315: 29-32, 2001.
23. House SJ, Potier M, Bisaillon J, Singer HA, and Trebak M. The non-excitable smooth muscle: calcium signaling and phenotypic switching during vascular disease. Pflugers Archiv : European journal of physiology 456: 769-785, 2008.
91 24. Hubbell MC, Semotiuk AJ, Thorpe RB, Adeoye OO, Butler SM, Williams JM, Khorram O, and Pearce WJ. Chronic hypoxia and VEGF differentially modulate abundance and organization of myosin heavy chain isoforms in fetal and adult ovine arteries. Am J Physiol Cell Physiol 303: C1090-1103, 2012.
25. Jackman K, and Iadecola C. Neurovascular regulation in the ischemic brain. Antioxidants & redox signaling 22: 149-160, 2015.
26. Johnston MV, Trescher WH, Ishida A, and Nakajima W. Neurobiology of hypoxic-ischemic injury in the developing brain. Pediatr Res 49: 735-741, 2001.
27. Kelly BA, Lewandowski AJ, Worton SA, Davis EF, Lazdam M, Francis J, Neubauer S, Lucas A, Singhal A, and Leeson P. Antenatal glucocorticoid exposure and long-term alterations in aortic function and glucose metabolism. Pediatrics 129: e1282-1290, 2012.
28. Khorram O, Ghazi R, Chuang TD, Han G, Naghi J, Ni Y, and Pearce WJ. Excess maternal glucocorticoids in response to in utero undernutrition inhibit offspring angiogenesis. Reprod Sci 21: 601-611, 2014.
29. Krugers HJ, Maslam S, Korf J, Joels M, and Holsboer F. The corticosterone synthesis inhibitor metyrapone prevents hypoxia/ischemia-induced loss of synaptic function in the rat hippocampus. Stroke; a journal of cerebral circulation 31: 1162- 1172, 2000.
30. Laugesaar R, Kolk A, Tomberg T, Metsvaht T, Lintrop M, Varendi H, and Talvik T. Acutely and retrospectively diagnosed perinatal stroke: a population-based study. Stroke; a journal of cerebral circulation 38: 2234-2240, 2007.
31. Leret ML, Rua C, Garcia-Montojo M, Lecumberri M, and Gonzalez JC. Influence of metyrapone treatment during pregnancy on the development and maturation of brain monoaminergic systems in the rat. Acta physiologica (Oxford, England) 197: 333-340, 2009.
32. Levine S. Regulation of the hypothalamic-pituitary-adrenal axis in the neonatal rat: the role of maternal behavior. Neurotoxicity research 4: 557-564, 2002.
33. Liu Y, Silverstein FS, Skoff R, and Barks JD. Hypoxic-ischemic oligodendroglial injury in neonatal rat brain. Pediatr Res 51: 25-33, 2002.
34. Lynch JK, and Nelson KB. Epidemiology of perinatal stroke. Current opinion in pediatrics 13: 499-505, 2001.
35. Matchkov VV, Kudryavtseva O, and Aalkjaer C. Intracellular Ca(2)(+) signalling and phenotype of vascular smooth muscle cells. Basic Clin Pharmacol Toxicol 110: 42-48, 2012.
92 36. Nunez J. Sex and steroid hormones in early brain injury. Reviews in endocrine & metabolic disorders 13: 173-186, 2012.
37. O'Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, and Howells DW. 1,026 experimental treatments in acute stroke. Annals of neurology 59: 467-477, 2006.
38. Owens GK, Kumar MS, and Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiological reviews 84: 767-801, 2004.
39. Qiao M, Meng S, Scobie K, Foniok T, and Tuor UI. Magnetic resonance imaging of differential gray versus white matter injury following a mild or moderate hypoxic- ischemic insult in neonatal rats. Neurosci Lett 368: 332-336, 2004.
40. Rice JE, 3rd, Vannucci RC, and Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Annals of neurology 9: 131-141, 1981.
41. Roghair RD, Segar JL, Sharma RV, Zimmerman MC, Jagadeesha DK, Segar EM, Scholz TD, and Lamb FS. Newborn lamb coronary artery reactivity is programmed by early gestation dexamethasone before the onset of systemic hypertension. Am J Physiol Regul Integr Comp Physiol 289: R1169-1176, 2005.
42. Smith JT, and Waddell BJ. Increased fetal glucocorticoid exposure delays puberty onset in postnatal life. Endocrinology 141: 2422-2428, 2000.
43. Suwanjang W, Holmstrom KM, Chetsawang B, and Abramov AY. Glucocorticoids reduce intracellular calcium concentration and protects neurons against glutamate toxicity. Cell calcium 53: 256-263, 2013.
44. Trainer PJ. Corticosteroids and pregnancy. Seminars in reproductive medicine 20: 375-380, 2002.
45. Vutskits L. Cerebral blood flow in the neonate. Paediatric anaesthesia 24: 22-29, 2014.
46. Wamhoff BR, Bowles DK, and Owens GK. Excitation-transcription coupling in arterial smooth muscle. Circ Res 98: 868-878, 2006.
47. Wang S, Wu EX, Cai K, Lau HF, Cheung PT, and Khong PL. Mild hypoxic- ischemic injury in the neonatal rat brain: longitudinal evaluation of white matter using diffusion tensor MR imaging. AJNR Am J Neuroradiol 30: 1907-1913, 2009.
48. Wang S, Wu EX, Tam CN, Lau HF, Cheung PT, and Khong PL. Characterization of white matter injury in a hypoxic-ischemic neonatal rat model by diffusion tensor MRI. Stroke; a journal of cerebral circulation 39: 2348-2353, 2008.
93 49. Wu YW, Lynch JK, and Nelson KB. Perinatal arterial stroke: understanding mechanisms and outcomes. Seminars in neurology 25: 424-434, 2005.
50. Zar JH. Biostatistical analysis. Upper Saddle River, N.J.: Prentice Hall, 1999.
94 CHAPTER FOUR
VALIDATION OF A MODEL OF MILD ISCHEMIA
Introduction
The following chapter describes experimental work that is still in progress, and does not constitute a completed project. Nevertheless, this section is a culmination of the efforts that have been put into validating our model of mild hypoxia-ischemia, as seen in
Chapter Three. As indicated, we have developed a model of mild hypoxia-ischemia that results in global edema at 24 hours post-injury and is detectable by MRI but that does not result in a consolidated infarct or lesion, unlike more classic models which result in an average of 30-40% brain infarction. In the model, postnatal day 9 or 10 pups undergo unilateral carotid ligation and a 24-hour recovery period, followed by 90 minutes of exposure to 8% hypoxia. Due to the mildness of the injury, it was necessary to perform a number of validation experiments including histology, neurobehavioral studies, and MRI analysis. Collectively, these results indicate that we have developed a model of mild hypoxia-ischemia that does not result in a typical infarct pattern. Yet, despite this, we still see a number of significant vascular effects such as changes to vessel calcium sensitivity and structure. We conclude that the vasculature appears to be a very sensitive indicator of ischemic damage, warranting future analysis.
Materials and Methods
In order to provide a measurement of infarct levels, a separate set of experimental animals (n=5 per group; procedures as described in Chapter 3) were transcardially perfused with 4% paraformaldehyde using a positive pressure system. Brains were then
95 removed and stored in fixative overnight at 4 ̊ C before being rinsed three times with PBS
(pH 7.4) and then transferred to a 30% sucrose solution with PBS. Once saturated, the brains were then cut into 2 mm slices and frozen at -80 ̊ C. Brain sections were later cut at
25um using a fixed-blade cryostat (Leica CM3050S). Sections were then taken for
histological analysis using the protocols described below.
FluoroJade-C
FluoroJade C was purchased from Histo-Chem (30mg 1FJC; Jefferson, Arkansas)
and a stock solution of 0.01% was prepared ahead of time and stored at 4 C.̊ A
standardized procedure was then performed in which the brain sections were first
rehydrated by use of a basic alcohol solution (1% NaOH in 80% EtOH), 70% EtOH,
followed by distilled water. Slides were then immersed into a 0.06% potassium
permanganate (KMnO4) solution for 5 minutes in order to reduce background signal, after which they were exposed to a diluted FJC solution (0.0001% FJC in 0.1% acetic acid) for 10 minutes on an autoshaker (0.60 min-1). The diluted FJC solution was used within one hour of preparation. Slides were then rinsed thrice in DI water, dried at room temperature, heated at 50 C̊ for 5 minutes, and then dipped in Histoclear. All slides were cover-slipped with Omnimount for imaging. All images were taken with a Keyence BZ-
X700 All in One Fluorescence Microscope. Tiled images were taken at 10X magnification and then combined with a 30% overlap using the BZ-X700 Analyzer software.
96 C-fos
Anti-c-Fos (Ab-2) antibody was purchased from EMD Millipore (PC05-100UG;
EMD Millipore, Temecula, CA). Slides stored at -20 C̊ were warmed at 37 C̊ for 30
minutes, washed thrice in PBS with 0.3% Triton X-100 (PBS-T, pH 7.45) for 10 min each. Endogenous peroxidases were quenched with 0.09% H2O2 in 1X PBS-T for 30
minutes. Slides were washed again before undergoing blocking for 1 hour (2% normal
goat serum and 1% bovine serum albumin in 1X PBS with 0.01% Triton X-100). The
slides were then incubated with primary antibody at a titer of 1:20 (5 µg/ml) overnight at
room temperature on a rotator. The next day, slides were washed again in PBS (thrice for
15 minutes each), and then incubated in secondary antibody (1:300 Dylight 633 goat-anti
rabbit) for 1.5 hours in the dark. The slides were then washed again in PBS-T twice for
10 min followed by 10 min in 1X PBS before being dried. Vectashield Hard Set
mounting media with DAPI was then applied and the slides were cover-slipped.
Glial Fibrillary Acidic Protein (GFAP)
Anti-Glial Fibrillary Acidic Protein (GFAP) Antibody was purchased from EMD
Millipore (MAB3402; Temecula, CA). Slides were first warmed at room temperature and
then 5 minutes at 37 C.̊ Slides were washed in 1X PBS (pH 7.45) thrice for 15 minutes
before being blocked (2% bovine serum albumin in PBS) for 1.5 hours. They were then
incubated overnight on a rocker with primary antibody (1:1000 titer). The next day, slides
were quickly washed in PBS followed by thrice in PBS for 15 minutes each. Slides were
incubated for 1.5 hours in the dark (1:300 titer). They were then washed thrice for 10
97 minutes each before being dried and then set with Vectashield hard set mounting media and cover-slipped. All slides were stored at -4 C̊ until imaged.
Ionized Calcium-Binding Adapter (IBA-1) Protein
Anti ionized calcium-binding adapter molecule (IBA-1) rabbit antibody was purchased from Wako Pure Chemical Industries (019-19741; Richmond, VA). Slides were first warmed up at 37 C̊ for 5 minutes before being washed thrice in 1X PBS (pH
7.45) for 15 minutes each. Slides were then exposed to 1% sodium dodecyl sulfate (SDS) for 10 minutes at room temperature. Excess SDS was removed and the slides were then washed thrice with PBS for 10 minutes each. Blocking was performed with 2% bovine serum albumin in 1X PBS at room temperature using a rocker. They were then incubated overnight in primary antibody (1:400 titer) on a rotator. The next day, slides were washed thrice in PBS with 0.1% Tween for 15 minutes each. Slides were then incubated in the dark with secondary antibody (1:150) for 2 hours. Slides were then washed twice with
PBS-T followed by 10 minutes in 1X PBS. Slides were sealed with Vectashield Hard Set
Mounting Media and then cover-slipped. As before, all slides were stored in the dark at 4
C in refrigerator.
Magnetic Resonance Imaging (MRI)
Magnetic Resonance Imaging (MRI) scanning was performed by the laboratory of
Dr. Andre Obenaus. Due to the ongoing nature of this project, the details of the methods used are not yet available.
98 Results & Discussion
We have developed a model of mild hypoxia-ischemia, where surgery is
performed on P9/P10 pups followed by a 24 hour recovery period with their dam and
then 90 minutes of exposure to hypoxia (8% oxygen). This model of mild ischemia is
characterized by changes to middle cerebral artery (MCA) structure and function, as measured by vessel bath myography and confocal microscopy, all of which occur without a consolidated infarct, as measured by FJ-C, IBA-1, c-fos, and cresyl violet staining.
Characterization of the model of mild hypoxia-ischemia revealed evidence of increased
ventricular edema, cellular stress/injury, and neurobehavioral differences. This model has
provided us a unique opportunity to study vascular responses to injury in the absence of
large infarcts, and supports the conclusion that the vasculature may be uniquely sensitive
to ischemic injury and may serve as a first indicator, even before major neuronal
involvement begins.
In order to fully characterize our model of mild hypoxia-ischemia, we employed a
number of methods including TTC, H&E, cresyl violet, FluoroJade-C, and MRI. The first
method we used was TTC staining, which tests the metabolic activity of tissues by
measuring dehydrogenase activity (3). Using this method, we demonstrated the often
punctate and variable nature of the hypoxic-ischemic damage in the neonatal brain (See
Figure 1). However, for a number of reasons, TTC did not seem to be an ideal method.
Previous labs have documented that tissue can be metabolically stunned and later
recover, leading to false negative results when staining with TTC (4). For these reasons,
we decided to pursue alternative methods of injury quantification, including histology
and MRI.
99
Figure 1. Measurements of Cerebral Infarct in a Model of Mild Ischemia. Initial attempts to quantify infarct volumes in this model of mild ischemia used TTC staining. Unilateral carotid ligation + 8% hypoxia resulted in cerebral ischemia. Representative frontal brain slices from four P12 Control-fed pups stained with 2% TTC showing infarcts after carotid ligation followed by either 90 minutes, A + B, or 120 minutes, C + D. Infarct percentages were analyzed in Photoshop and were comparable between treatment groups (27.6% vs 27.9%). In particular, please note the punctate and diffuse quality of the ischemic insult. However, due to concerns about the reliability and accuracy of the TTC method, further experiments and validation were deemed necessary.
100 There are many histological techniques that are useful for determining brain
injury: two of the most common ones are Cresyl Violet (CV), and hematoxylin and eosin
(H&E). CV stains the Nissl substance in neurons and can be used in conjunction with
light microscopy techniques to examine any gross histological changes that can occur
following neuronal damage. Preliminary analysis using sections of our model did not
reveal any obvious sections of damage (data not shown). H&E staining yielded similar
results, and it was difficult to distinguish between healthy and injured tissue (data not
shown). We did see evidence that our ischemic pups show signs of neurobehavioral differences (see Figure 2).
101
Figure 2. Behavior Data from Sham vs Ischemia Animals in mild Hypoxia-Ischemia: Righting Reflex, Geotaxis Reflex, and Open Field Exploration. Behavioral testing was performed on a number of pups from both Ischemia and Sham groups in order to determine neurological outcomes. It was found that the geotaxis reflex, but not the righting reflex or the open field test, were impaired post-injury. This result indicated that the model of mild hypoxia-ischemia, despite the lack of a consolidated infarct, still produces neurobehavioral differences.
102 From a functional whole-animal perspective, this would indicate that there is ischemic injury. However, it became clear that we needed more nuanced techniques would be required in order to validate our model. Next, we tried FluoroJade C (FJ-C)
(See Figure 3). FJ-C is an acidic stain derived from fluorescein. The mechanism is currently unknown, but it is commonly used to stain degenerating neurons ex vivo and has been used in neonatal hypoxia-ischemia studies (1). We are still finishing our analysis, but thus far have not seen evidence of a consolidated infarct in our model of mild HIE.
103
Figure 3. 4-Way Histological Analysis with GFAP, IBA-1, c-fos, and FJC of Sham vs Ischemia Pup Brains. Preliminary analysis of serial cross sections of pup brains from Sham and Ischemia animals taken at P12, 24-hours post injury (n=1 for both Sham and Ischemia). 4-Way histological staining with c-fos, GFAP, FJC, and IBA-1 was consistent with that of a mild hypoxia-ischemia injury and indicated the presence of widespread cellular stress and/or injury. A more complete quantitative analysis will be performed once the staining has been completed all treatment groups.
104 In addition to these vital dye staining techniques, there are also many potential
biomarkers for neonatal brain damage including markers of immune system activity, cell
death, or neuronal differentiation (19). We decided to investigate three of these: c-fos,
GFAP, and IBA-1. First, c-fos is a proto-oncogene from the Fos family of transcription factors. It can be considered a marker of cellular stress akin to heat shock protein (HSP)
70. It has been shown to be strongly activated following HIE, with c-fos potentially playing a neuroprotective role in certain regions such as the hippocampus (10, 19). Second,
GFAP is an interfilament protein expressed by many cells of the central nervous system
(CNS) including astrocytes and ependymal cells. Although it does not seem to be commonly stained for in the pup, it may sometimes be used as a biomarker of HIE (8) and is often used as a marker for neuronal damage in adult rats. We do not yet know what
pattern of staining we will see with GFAP, though the cells may be swollen near the
ventricles. A third possible marker is ionized calcium-binding adaptive molecule (IBA-
1), also known as allograft inflammatory factor 1 (AIF-1). It is a protein expressed in
microglia and macrophages following a neuronal injury such as ischemia and can be
found in activated macrophages (20). As there is frequently up-regulation of the immune
system and specifically evidence of microglial upregulation during HIE (16), we should see some activity after IBA-1 staining. For all of these methods, we are still waiting on our completed results (See Figure 3).
Another method that we have made use of is magnetic resonance imaging (MRI).
MRI is a well-established and sensitive method for characterizing early types of stroke and is a key diagnostic tool in the clinic (9). Because we have suspected that our model was mild, we have turned to increasingly more sensitive methods of injury quantification.
105 While we are still waiting on a complete analysis of our MRI results, preliminary
evidence from Sham and Ischemic animals suggests that our injury is primarily a global
one, affecting both the contralateral and ipsilateral sides, and resulting in edema and
ventricular swelling (see Figure 4). Several studies have looked at models of milder HIE
(17, 22, 28, 29). In general, there is relative agreement that the intensity of the insult can be modified by factors such as the duration of the hypoxic insult. Ninety minutes is usually considered to be the threshold to produce a lesion. In these models of mild ischemia, some of the most common features are oligodendrocyte injury, resulting in demylelination, predominantly white matter injury, and altered diffusion tensor MR imaging. In our case, we had originally hypothesized that exposing the pups to hypoxia at the 24 hour mark would increase infarct levels due to the neuroprotective effects of isoflurane. Instead, we now conclude that the long recovery period post-surgery was an
important factor in determining the severity of the insult, and that our model most closely
resembles that produced by a shorter dosage of hypoxia. Although we did not perform
myelin basic protein (MBP) immunostaining, we would expect a similar pattern as to
what has been previously reported (see references above).
106
Figure 4. Comparison of T2 and ADC in Sham vs Ischemia. MRI scanning (T2 and ADC) was performed on Sham and Ischemia ex vivo pup brains in order to characterize the injury (n=5 per group). It was found that mild Ischemia compared with Sham significantly increased MRI T2, and an analysis of the slope between T2 and ADC were found to be significantly different. This finding is consistent with cellular swelling and edema.
107 Clinically, perinatal stroke frequently results in injury to the brain stem, thalamus,
putamen, peri-Rolandic cerebral cortex resulting in motor impairments characteristic with
cerebral palsy (13). Full term infants are more sensitive to grey matter injury while
premature infants may be more sensitive to white matter injury. Motor function
impairment seems to be a common symptom with about ½ to ⅔ of infants displaying
such behavior. Grey matter injury may be more common in full term infants, whereas
diffuse injury is more applicable to premature newborns (9). Much of what we know
about neonatal ischemic was adapted from research in adults. However, neonatal stroke is
not the same as adult stroke, showing a number of differences. There are specific
vulnerabilities of the neonatal brain which should be taken into account, including
increased susceptibility to excitotoxicity, free radical injury, oxidative stress. The
neonatal brain also shows a greater tendency toward apoptotic cell death, as well as
sensitivity to oligodendrocyte injury (12). With respect to the vasculature, blood brain
barrier (BBB) function and neuronal proteases may also be compromised during HIE (2).
Angiogenesis after stroke is also thought to be important. Finally, endothelial progenitor cells (EPC’s) may play an important role in HIE.
Studying the effects of GC on development in humans is complicated and likely involves both genomic and non-genomic actions. GCs may increase neuronal stem progenitor cells (NSPC) proliferation. NSPCs play a key role in development and have many nuclear receptors, including GR (5). GR expression increases and peaks at around
P15 (14). A number of studies have looked specifically at the effects of glucocorticoids on neonatal stroke. In a 1995 study, treatment of synthetic glucocorticoid dexamethasone
4 hours prior to infarct resulted in neuroprotection in an HIE model (25, 26). Two stroke
108 studies reported neuroprotection with administration of MET 30 minutes (24) and 1 hour post-hypoxia (6). Infarcts were worsened when MET was administered post-insult (23). A
notable difference between these studies and the present one is that it involves acute
administration immediately before or after stroke. Given this evidence, it seems likely
that HPA axis activity has an influence on stroke outcome (30).
With respect to antenatal glucocorticoids, some studies have been performed,
most often in order to investigate the ramifications of glucocorticoid administered before
birth in order to prepare the premature fetus’s lungs for birth. GCs confer significant
benefits to infant survival and lung maturation (5), despite the potential for long-term
health problems. With respect to MET treatment before birth, there are not many studies
that have looked at this combination and the precise effects of glucocorticoid inhibition
during gestation are unknown. Our work has indicated a mild effect of gestational
metyrapone on weight gain, with and without ischemia (see Figure 5). The effects of
MET on the brain are not very well characterized, but may include altered dopaminergic
and serotoninergic pathways. Leret et al found that Metyrapone administered from E0 to
E17 of gestation resulted in alterations to dopamine and serotonin signaling that persisted
to at least P90 (15). While the timing and method of dosage was different (E10-E21, 0.5
mg/ml via drinking water vs E0-E17, 45 mg/kg via i.p. injection), these results
nevertheless indicate that glucocorticoid inhibition, like glucocorticoid excess, may also
result in fetal programming. This is in agreement with our findings from Chapter 3.
109
Figure 5. Body Weights of Pups at P11/12 Post-Treatment. Body weights of rat pups from all four treatment groups were measured immediately prior to sacrifice on postnatal days 11 and 12. Compared with Sham animals, the Ischemia group showed a significant decrease in body weight. By comparison, MET-treated pups did not show any significant weight loss post-Ischemia. MET-treated animals also exhibited a slight but significant weight increase compared with both Sham and Ischemia pups. This data indicates that gestational Metyrapone is protective against Ischemia-induced weight loss and may contribute to increased body weight at postnatal days 11 and 12.
110 The specific effects of GC influences on the developing vasculature are understudied. Some papers, in their quest to understand the dramatic effects of GCs on lung maturation (especially with regards to surfactant production) have looked at the effects that GCs may have in the vasculature of the lungs (7, 11). Nevertheless, little is known about the vasculature in other organ systems such as the brain. One study from
2008 found that fetal cerebral blood flow in lambs was decreased 4 hours after two intramuscular betamethasone injections, indicating a vasoconstrictor effect by GCs (18).
Another study using a similar model also reported decreased middle cerebral artery vascular contractility in adult sheep that were exposed to two doses of prenatal betamethasone(18). These two studies combined suggest that GC exposure early in life may affect adults differently than fetuses, with a tendency toward increased contractility as a fetus vs decreased contractility as an adult. A third paper also suggested that GCs may have effects on the cerebral vasculature. In a model of periventricular- intraventricular hemorrhage, prenatal glucocorticoid administration was shown to significantly reduce the risk of hemorrhage and mortality rate in preterm infants (21).
Work by Vinukonda et al in a germinal matrix hemorrhage model has shown that prenatal
GC exposure may reduce angiogenesis via VEGF, increase TGF-ß levels, and suppress endothelial proliferation (27). The authors concluded that reduced vascular density may be a factor in the reduced risk following GC therapy. Although this work has been done with respect to a model of cerebral hemorrhage, it nevertheless gives us some insight into the status of the vasculature following GC exposure and the possibility that GCs may have a stabilizing effect on the developing vasculature. Though it requires some speculation, the inhibition of glucocorticoids during pregnancy via metyrapone would
111 then be expected to act in an opposite way, perhaps favoring a more angiogenic phenotype and allowing for more collateral circulation depending on the brain region.
Conclusions
In conclusion, we have presented preliminary evidence that our model of mild hypoxia-ischemia may be a novel technique for examining vascular injury in the absence of a consolidated ischemic infarct. To support this claim, we have presented evidence from neurobehavioral studies, multiple histological stains, and MRI data. It should be noted, however, that this work does not yet include our Metyrapone groups, either
Ischemia+MET or Sham+MET, and that these experiments are currently underway.
Likewise, in the context of the greater dissertation and project, it will also be necessary to validate the model with respect to maternal food restriction.
112 References
1. Aggarwal M, Burnsed J, Martin LJ, Northington FJ, and Zhang J. Imaging neurodegeneration in the mouse hippocampus after neonatal hypoxia-ischemia using oscillating gradient diffusion MRI. Magnetic resonance in medicine 72: 829-840, 2014.
2. Arai K, Jin G, Navaratna D, and Lo EH. Brain angiogenesis in developmental and pathological processes: neurovascular injury and angiogenic recovery after stroke. The FEBS journal 276: 4644-4652, 2009.
3. Bederson JB, Pitts LH, Germano SM, Nishimura MC, Davis RL, and Bartkowski HM. Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke; a journal of cerebral circulation 17: 1304-1308, 1986.
4. Benedek A, Moricz K, Juranyi Z, Gigler G, Levay G, Harsing LG, Jr., Matyus P, Szenasi G, and Albert M. Use of TTC staining for the evaluation of tissue injury in the early phases of reperfusion after focal cerebral ischemia in rats. Brain Res 1116: 159-165, 2006.
5. Carson R, Monaghan-Nichols AP, DeFranco DB, and Rudine AC. Effects of antenatal glucocorticoids on the developing brain. Steroids 114: 25-32, 2016.
6. Charles MS, Ostrowski RP, Manaenko A, Duris K, Zhang JH, and Tang J. Role of the pituitary-adrenal axis in granulocyte-colony stimulating factor-induced neuroprotection against hypoxia-ischemia in neonatal rats. Neurobiology of disease 47: 29-37, 2012.
7. Deruelle P, Houfflin-Debarge V, Magnenant E, Jaillard S, Riou Y, Puech F, and Storme L. Effects of antenatal glucocorticoids on pulmonary vascular reactivity in the ovine fetus. Am J Obstet Gynecol 189: 208-215, 2003.
8. Douglas-Escobar M, and Weiss MD. Biomarkers of hypoxic-ischemic encephalopathy in newborns. Front Neurol 3: 144, 2012.
9. Fernandez-Lopez D, Natarajan N, Ashwal S, and Vexler ZS. Mechanisms of perinatal arterial ischemic stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 34: 921-932, 2014.
10. Ginet V, Puyal J, Magnin G, Clarke PG, and Truttmann AC. Limited role of the c-Jun N-terminal kinase pathway in a neonatal rat model of cerebral hypoxia- ischemia. J Neurochem 108: 552-562, 2009.
11. Goldsmith AM, Hershenson MB, Wolbert MP, and Bentley JK. Regulation of airway smooth muscle alpha-actin expression by glucocorticoids. American journal of physiology Lung cellular and molecular physiology 292: L99-L106, 2007.
113 12. Grow J, and Barks JD. Pathogenesis of hypoxic-ischemic cerebral injury in the term infant: current concepts. Clinics in perinatology 29: 585-602, v, 2002.
13. Johnston MV, Trescher WH, Ishida A, and Nakajima W. Neurobiology of hypoxic-ischemic injury in the developing brain. Pediatr Res 49: 735-741, 2001.
14. Lee BH, Wen TC, Rogido M, and Sola A. Glucocorticoid receptor expression in the cortex of the neonatal rat brain with and without focal cerebral ischemia. Neonatology 91: 12-19, 2007.
15. Leret ML, Rua C, Garcia-Montojo M, Lecumberri M, and Gonzalez JC. Influence of metyrapone treatment during pregnancy on the development and maturation of brain monoaminergic systems in the rat. Acta physiologica (Oxford, England) 197: 333-340, 2009.
16. Liu F, and McCullough LD. Inflammatory responses in hypoxic ischemic encephalopathy. Acta pharmacologica Sinica 34: 1121-1130, 2013.
17. Liu Y, Silverstein FS, Skoff R, and Barks JD. Hypoxic-ischemic oligodendroglial injury in neonatal rat brain. Pediatr Res 51: 25-33, 2002.
18. McCallum J, Smith N, Schwab M, Coksaygan T, Reinhardt B, Nathanielsz P, and Richardson BS. Effects of antenatal glucocorticoids on cerebral substrate metabolism in the preterm ovine fetus. Am J Obstet Gynecol 198: 105.e101-109, 2008.
19. Ness JM, Harvey CR, Washington JD, Roth KA, Carroll SL, and Zhang J. Differential activation of c-fos and caspase-3 in hippocampal neuron subpopulations following neonatal hypoxia-ischemia. Journal of neuroscience research 86: 1115- 1124, 2008.
20. Patel SD, Brennan G, Brazin J, Ciardiello AJ, Silver RB, and Vannucci SJ. Mast cell isolation from the immature rat brain. Developmental neuroscience 35: 265-271, 2013.
21. Perlman JM. The relationship between systemic hemodynamic perturbations and periventricular-intraventricular hemorrhage--a historical perspective. Seminars in pediatric neurology 16: 191-199, 2009.
22. Qiao M, Meng S, Scobie K, Foniok T, and Tuor UI. Magnetic resonance imaging of differential gray versus white matter injury following a mild or moderate hypoxic- ischemic insult in neonatal rats. Neurosci Lett 368: 332-336, 2004.
23. Risedal A, Nordborg C, and Johansson BB. Infarct volume and functional outcome after pre- and postoperative administration of metyrapone, a steroid synthesis inhibitor, in focal brain ischemia in the rat. European journal of neurology 6: 481-486, 1999.
114 24. SmithSwintosky VL, Pettigrew LC, Sapolsky RM, Phares C, Craddock SD, Brooke SM, and Mattson MP. Metyrapone, an inhibitor of glucocorticoid production, reduces brain injury induced by focal and global ischemia and seizures. Journal of Cerebral Blood Flow and Metabolism 16: 585-598, 1996.
25. Tuor UI. Dexamethasone and the prevention of neonatal hypoxic-ischemic brain damage. Annals of the New York Academy of Sciences 765: 179-195; discussion 196- 177, 1995.
26. Tuor UI. Glucocorticoids and the prevention of hypoxic-ischemic brain damage. Neurosci Biobehav Rev 21: 175-179, 1997.
27. Vinukonda G, Dummula K, Malik S, Hu F, Thompson CI, Csiszar A, Ungvari Z, and Ballabh P. Effect of prenatal glucocorticoids on cerebral vasculature of the developing brain. Stroke; a journal of cerebral circulation 41: 1766-1773, 2010.
28. Wang S, Wu EX, Cai K, Lau HF, Cheung PT, and Khong PL. Mild hypoxic- ischemic injury in the neonatal rat brain: longitudinal evaluation of white matter using diffusion tensor MR imaging. AJNR Am J Neuroradiol 30: 1907-1913, 2009.
29. Wang S, Wu EX, Tam CN, Lau HF, Cheung PT, and Khong PL. Characterization of white matter injury in a hypoxic-ischemic neonatal rat model by diffusion tensor MRI. Stroke; a journal of cerebral circulation 39: 2348-2353, 2008.
30. Xiong F, and Zhang L. Role of the hypothalamic-pituitary-adrenal axis in developmental programming of health and disease. Frontiers in neuroendocrinology 34: 27-46, 2013.
115 CHAPTER FIVE
CONCLUSIONS AND FUTURE DIRECTIONS
Maternal food restriction and other related pathologies such as intrauterine growth restriction can contribute to lifelong chronic health problems in adults. These conditions are all connected by adverse conditions during critical periods of development where key structural and functional decisions are made by the growing fetus in response to changing environmental conditions. These development differences then manifest as permanent physiological and anatomical changes which the fetus retains as they mature to adulthood. Of the systems effected by food restriction, this dissertation has primarily focused on changes related to the cerebrovasculature.
Chapter One introduced the general research topic and sought to provide some important background information about the state of the food restriction field. It introduced the concept of fetal programming, and how glucocorticoids may be at least partially responsible for food restriction-related anatomical changes. In it, we also explored the basics of cerebrovascular autoregulation, vascular anatomy, and smooth muscle physiology. We also spoke about the clinical impact that hypoxia-ischemia may have on an infant and some of the treatments currently available, as well as the gap in knowledge that this dissertation seeks to address.
In Chapter Two, we established that 50% maternal food restriction during Days
10 through 21 of gestation can produce significant changes to the cerebrovascular structure and function in 8-month old adult male offspring and that these vascular changes were detectable by our chosen methods of vessel bath myography and immunohistochemistry in combination with confocal microscopy. This paper emphasized
116 the importance of glucocorticoid-related mechanisms by showing that gestationally
administered Metyrapone, a glucocorticoid synthesis inhibitor, reversed some of the
effects of MFR, most notably vascular stiffness. These experiments set the stage to ask
questions about the cerebrovasculature at earlier time points, especially the neonatal
period. Mainly, how early do vascular related changes such altered passive compliance or
depressed contractility begin? Are these differences detectable during postnatal life? On
a more practical level, we also wanted to know what the programming effects of
Metyrapone alone would be, as well as to find out how much of the changes that we saw
at 8-months of age were due to the animal’s overall adiposity and metabolic changes,
which can also influence vascular health, and how much was due strictly to food-
restriction. To do so, it was necessary to examine a neonatal model, before these overt
signs of premature aging.
In Chapter Three, we began asking questions about the neonatal vasculature. In
order to begin a thorough analysis of food restricted arteries, however, it was first
necessary to establish a baseline of pup artery structure and function. To do so, we
developed a model of mild ischemia, the purpose of which was to provide an appropriate
vascular stressor by which to test the normal pup physiological response. Control Sham
and Ischemia pups were exposed to hypoxia 24-hours post-surgery. Likewise, given the importance of excess glucocorticoid exposure during pregnancy, it was also necessary to determine the effects of glucocorticoid inhibition during pregnancy as a control experiment. Hence, a second cohort of animals was also studied alongside the first Sham and Ischemia groups. Female pregnant rats were administered Metyrapone on Day 11 through 21 of gestation, after which time they were administered normal drinking water.
117 By adapting the methods of vessel-bath myography and immunohistochemistry combined with confocal microscopy to our pup arteries, we found that our model of mild ischemia appears to be a sufficient stressor to produce vascular structure and function changes, and that gestational Metyrapone again appears to mediate and oppose some of these ischemia- induced changes. This indicates that glucocorticoid status established well in advance of an ischemic insult during mid-late gestation can fundamentally alter the vascular response to ischemia during the neonatal period.
Chapter Four includes ongoing experimental work for establishing and validating our model of mild ischemia. Some of the methods used to quantify and assess the brain injury of our pups include MRI scanning techniques, histological stains, and neurobehavioral analysis. From our preliminary data, we believe that we have developed a model of mild ischemia that produces inflammation and cellular swelling without a consolidated infarct. Despite this mild infarct, we nonetheless established in Chapter
Three that even mild hypoxia-ischemia is capable of eliciting a vascular response, which may be a key finding for future work. A complete analysis with our Metyrapone treatment group is currently underway and should provide additional information about the nature of our model.
Future work will involve looking into the effects of programming via gestational metyrapone and maternal food restricted animals in addition to our current neonatal stroke model. Our lab would also like to build upon work started with our collaborators at
Harbor UCLA and explore the possibility of additional epigenetic mechanisms such the influence of miRNAs, especially miRNA 29C. In keeping with this theme, it would also be of interest to explore other ways by which food restriction may be acting, by delving
118 into DNA methylation studies or perhaps histone acetylation. Such work has been explored in other tissues and models, but a focus on the cerebrovasculature could yield some unique insights. Together, this body of work should advance our understanding of how prenatal programming acts to mediate future health outcomes as well as inform clinicians on how to best treat high risk pregnancies.
119