Brain and retinal macro- and microvasculature Li, Youhai
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Brain and retinal macro- and
microvasculature
Response to ischemic and hyperglycemic stress
Cover design & Layout: Youhai Li (Xueying Feng supplies the paintings) Printed by: IPSKAMP Printing
Youhai Li
Brain and retinal macro- and microvasculature
Response to ischemic and hyperglycemic stress
ISBN (printed): 978-94-034-0994-8 ISBM (digital): 978-94-034-0993-1
Copyright © Youhai Li, 2018
All rights reserved. No part of this publication maybe reproduced or transmitted in any form or by any means without permission of the author.
Brain and retinal macro- and
microvasculature
Response to ischemic and hyperglycemic stress
PhD thesis
to obtain the degree of PhD at the University of Groningen
on the authority of the Rector Magnificus Prof. E. Sterken
and in accordance with
the decision by the College of Deans.
This thesis will be defended in public on Monday 15 October 2018 at 9.00 hours
by
Youhai Li
born on 9 October 1984Prof. Dr. Han Moshage Prof. Dr. Dr. Lothar Schilling
Co-supervisor
Dr. Jan Kamps
Assessment committee
Prof. Dr. G. (Ingrid) Molema Prof. Dr. Jan-Luuk Hillebrands Prof. Dr. Hans-Peter Hammes
Dr. Jing Wang
Dayang Erna Zulaikha
Chapter 1
General introduction
Chapter 2
Enhancement of bradykinin-induced relaxation by focal brain ischemia in the rat middle cerebral artery: Receptor expression upregulation and activation of multiple pathways
Chapter 3
A novel method to isolate retinal and brain microvessels from individual rats: Microscopic and molecular biological characterization and application in hyperglycemic animals
Chapter 4
Comparative transcriptome analysis of inner retinal barrier and blood-brain barrier in rats
Chapter 5
Responses of retinal and brain microvasculature to diabetes revealed by global expression profiling Chapter 6 General discussion Appendices Summary Nederlandse samenvatting List of publications Acknowledgments
CHAPTER 1
General Introduction
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The architecture of brain and retinal vascular system
The vertebrate central nervous system (CNS) consists of the brain, retina and spinal cord. The brain is one of the most highly perfused and most energetically expensive organs across mammalian species (1). The average adult human brain accounts for only about 2% of the body weight and occupies approximately 1200 cm3, yet it receives about 20% of the cardiac output and accounts for one fifth of the body’s total energy consumption under resting conditions (2). Overall, the blood vessels throughout the body can be divided into two classes according to their size: the macrovessels and the microvessels. The macrovessels consist of arteries, big arterioles, big venules and veins that usually can be seen with the naked eye. The microvessels consist of pre-capillary arterioles, capillaries and post-capillary venules that can only be observed under the microscope. The blood delivery to neuronal tissues via the CNS vascular system has two key functions:
1) Controlling the cerebral blood flow (CBF) and propelling the oxygenated blood forward into smaller arterioles, which is mainly achieved at the level of macrovessels;
2) Modulating the exchange of nutrients, hormones, ions and metabolites between circulating blood and parenchymal tissue, which takes place at the level of microvessels.
Delivering oxygenated blood to brain tissue
Oxygenated blood is delivered into the brain tissue through two pairs of large arteries: (i) The right and left internal carotid arteries (ICA), which divide into the middle cerebral artery (MCA), the anterior cerebral artery (ACA) and the posterior cerebral arteries (PCA); (ii) The right and left vertebral arteries merge into the basilar artery at the base of the brain stem which then connects to the PCA by the posterior communicating artery (PComA). These arteries are further separated into pial branches (smaller arteries and arterioles), that travel along the surface of the brain across the subarachnoid space (figure 1B). The oxygenated blood is carried into the brain parenchyma via the radially penetrating arterioles that are located in the Virchow-Robin space and are bathed with cerebrospinal fluid (3). In contrast to peripheral
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microcirculation, the brain terminal vascular networks have an unique and protective structure, the blood-brain barrier (BBB), the structural correlate of which is found in the microvascular endothelial cells (4). The exchange of ions, nutrients (e.g., glucose and amino acids) and metabolic waste products between circulating blood and brain tissue is tightly regulated within the cerebral microvessel by the BBB, to protect the neural tissue from fluctuations in blood composition and maintain the brain microenvironment homeostasis (5, 6).Figure 1. Representative images showing rat brain vasculature.
A) Image of the base of the rat brain. The right and left internal carotid arteries (ICA, removed
during brain dissection) divide at the base of the brain, giving rise to the anterior cerebral artery (ACA), the middle cerebral artery (MCA) and the posterior cerebral artery (PCA). Two vertebral arteries (removed during brain dissection) merge into the basilar artery (BA), which is connected to the PCA by the posterior communicating artery (PComA). Blue represents the right side of the brain, while black represents left.
B) Microscopic image of the rat brain superficial vessels. Pial arteries travel among the surface
of brain and divided into smaller branches and arterioles.
C) Image of isolated capillary fractions stained with haemotoxylin and eosin (H&E). Nuclei
of endothelial cells and pericytes are clearly displayed.
Delivering oxygenated blood to retinal tissue
Philosophers defined the eye as a window to the soul. Scientists have struggled to investigate the structural and functional associations between eye and brain (7-9). During embryonic development, the vertebrate retina, optic nerve and iris originate from the forebrain neuroectoderm (10). The optic nerve carries the ganglion cell axons to the geniculate nucleus in the thalamus and the superior colliculus in the midbrain,
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from which the signals are further sent to brain cortex for visual processing. Similar to brain, most of the energy consumed in retina comes from oxidative metabolism coupled to ATP synthesis. Oxygenated blood is delivered into the retina and the optic nerve by several branches (e.g., the central retinal artery) from the ophthalmic artery (OA) that directly arises from the ICA in most mammals (11). The central retinal artery (CRA) penetrates into the retina through the bulbar part of the optic nerve at the optic disc and then divides into smaller arteries and arterioles in a radial course from the central to the peripheral area. Similar to the cerebral microvessels making up the BBB, the retinal microvessels also express a unique structure, the inner blood-retinal barrier (iBRB), to strictly regulate the paracellular and transcellular movement of molecules through the microvessel walls (12).Figure 2. Representative images showing rat inner retinal vasculature.
A) Phase contrast image of the rat retinal tissue, in which the microvessels are clearly
displayed. The freshly isolated retina was sandwiched between two glass coverslips.
B) Trypsin digested preparation of retinal vasculature stained by periodic acid-schiff (PAS).
(Image courtesy of Dr. Jing Wang).
C) Image of isolated capillary fractions stained with haemotoxylin and eosin (H&E). Nuclei
of endothelial cells and pericytes are clearly displayed
The vasomotor activities of cerebral arteries
The resistance to blood flow through peripheral vascular beds is influenced primarily by arteriolar vasomotor activities. However, in the brain, both arteries and arterioles contribute significantly to the resistance of cerebral blood flow, hence, both control the blood perfusion to the terminal vessels (13). The vessel wall is essentially composed of
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two types of cells: (i) a monolayer of endothelial cells that form a physical barrier known as BBB, which separates the brain tissue from the blood; and (ii) vascular smooth muscle cells (VSMCs) that surround the abluminal side of the endothelium and control the size of the macrovessel lumen through their vasomotor activities (relaxation or contraction). When VSMCs relax, the blood flow to the downstream vessels is increased. When VSMCs contract, the blood flow to the downstream vessels is reduced.Under physiological conditions, the endothelial cells of cerebral arteries are continuously exposed to circulating blood, while the VSMCs are separated from the circulating blood because of the BBB (14). Whether and how the circulating blood contribute to regulation of vasomotor activities of cerebral arteries has been unclear for a long time. In 1980, these open questions began to be resolved with the discovery of an endothelium-derived relaxing factor (EDRF) subsequently identified as nitric oxide (NO) (15). These discoveries formed the basis for the later award of the Nobel Prize in Physiology or Medicine warded jointly to Robert F. Furchgott, Louis J. Ignarro and Ferid Murad. Thereafter, increasing evidences demonstrated that vasoactive substances (e.g. bradykinin) in the circulating blood are able to activate their corresponding receptors (e.g. bradykinin receptor 1 and 2) on the surface of endothelial cells, followed by release of various endothelial-derived factors, e.g., NO, prostacyclin (PGI2) and endothelium-derived hyperpolarizing factor/s (EDHF) (16-18). These endothelial-derived factors can diffuse to the underlying VSMCs and induce different vasomotor activities of VSMCs via several endothelial-dependent mechanisms, such as NO-cGMP signaling pathway, PGI2-cAMP signaling pathway and EDHF-hyperpolarization signaling pathway (16, 17, 19, 20).
Under physiological conditions, the vasomotor activities of VSMCs in the CNS are endowed with powerful regulatory mechanisms. However, this precise control of CNS arteries can suffer damage due to impaired circulation. For example, many studies have shown that acute ischemic stroke not only affects brain parenchymal cells and the integrity of the BBB, but may also undermine the ability of the brain macrovasculature to maintain an appropriate level of CBF (21-23). The underlying molecular mechanisms are still far from elucidated. In chapter 2, using a rat acute cerebral ischemic model, we determined the changes of isometric force of MCA ring segments in response to bradykinin (BK) (figure 3). We aimed to explore whether transient cerebral ischemia
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impairs the vasomotor functions of the MCA to BK and to identify the underlying molecular mechanisms. Selective agonists and antagonists were used to identify the responsible receptor subtype underlying the BK-induced effect. Additionally, the possible underlying signaling pathways were investigated by several nitric oxide synthase (NOS) inhibitors and calcium-activated potassium channel (Kca) blockers.Figure 3 Outline of theisometric force measurements in rat cerebral arteries after acute
ischemic stroke. A) Schematic diagram of the rat middle cerebral artery (MCA) occlusion
using a silicone coated filament blocking the right MCA origin. CCA, common carotid artery; ECA, external carotid artery; PPA, pterygopalatine artery; ICA, internal carotid artery; PCA, posterior cerebral artery; MCA, middle cerebral artery; ACA, anterior cerebral artery. B) Image of representative rat brain (dorsal side) after 24 hours of right MCA occlusion. The occluded/right hemisphere displays a massive brain edema and enlarged pial vessels. C) Image of representative pial arteries that were carefully isolated for vasomotor function studies. D) Schematic diagram showing the set up for the isometric force measurements of the isolated cerebral artery ring segments (24). E) Screenshot of representative curve of the isometric force measurement on the rat isolated cerebral artery ring segments after acute ischemic stroke. The vertical axis represents the contractile force (in millinewton, mN). The horizontal axis represents time (in minutes).
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Blood-brain barrier and inner blood-retinal barrier
In 1885, Paul Ehrlich observed for the first time that water-soluble dyes injected into the peripheral circulation of experimental animals stained all the tissues of the body except the brain and spinal cord and attributed this phenomenon to a low affinity of CNS tissues for the dye (25). In 1900, the term Bluthirnschranke (blood-brain barrier, BBB) was pioneered by Lewandowsky to explain the limited permeation of potassium ferrocyanate into the brain parenchyma tissue (26). It wasn’t until 1913, when the iBRB was first observed in retina. Schnaudigel showed that the retinal microvessels expressed barrier characteristics similar to the BBB to limit the penetration of trypan blue into retina tissue (27). Further studies showed that interendothelial tight junctional complexes play a key role in preventing the dye molecules moving into neuronal tissues (28). The brain endothelial cells are characterized by the absence of fenestrations, high transendothelial electrical resistance and low rate of vesicular transport (transcytosis) (29-32). Furthermore, pericytes were found to embrace the abluminal surface of endothelium in microvessels (33). Many studies report that pericytes are indispensable for the development and maintenance of the integrity of the BBB and iBRB (34-36).
Diabetes mellitus
Globally, the prevalence of diabetes mellitus (DM) has risen dramatically over the past three decades (37). In 2030, the World Health Organization (WHO) projects that DM will be the 7th leading cause of death (http://www.who.int/mediacentre/en/). During the last two decades, many causal factors have been proposed for diabetic vascular complications, such as superoxide overproduction and NF-κB pathway activation in vascular cells (38, 39). Despite extensive research, up to now there are no effective therapies for preventing those vascular complications (40). This frustrating clinical report indicates that the molecular mechanisms of DM vascular complications are far from being revealed. Some researchers assumed that diabetic vascular complications may result from an imbalance between molecular mechanisms of injury and endogenous protective factors (41). This hypothesis provides some clues to pay attention to the healthy/resistant organs to DM in the same body (e.g., the brain).
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Disruption of the inner blood-retinal barrier in diabetes
Diabetic Retinopathy (DR) is a common microvascular complication in patients with diabetes and remains the leading cause of vision loss among the working-age people worldwide (42, 43). It is clinically defined as the presence of typical signs of retinal vascular damage (e.g., microaneurysms, cotton wool spots, exudates and hemorrhages) in individuals with long-lasting diabetes mellitus. Leakage of the iBRB is the fundamental feature of early DR (44, 45). Clinically, DR is usually divided into two major stages: the earlier stage of non-proliferative DR (NPDR) and the advanced stage of proliferative DR (PDR) (46). In diabetes patients, the leading cause of the impaired visual acuity is diabetic macular edema, which can occur at any stage of DR (47). Edema is characterized by exudative fluid and protein accumulation due to the disruption of iBRB in the macular region (Fig. 4) (48). Multiple biochemical mechanisms have been proposed to contribute to the pathogenesis of DR, such as hyperglycemia-induced oxidative stress, increased production of advanced glycation end products (AGEs) and activation of protein kinase C (43, 49).
Figure 4. Imaging methods to characterize vascular features of diabetic retinopathy. A
fundus image shows proliferative diabetic retinopathy with macular edema (upper panel). AN, arteriolar narrowing; HE, hard exudates; CWS, cotton wool spots; PRH, preretinal hemorrhage;
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NFH, nerve-fiber hemorrhage; VB, venous beading. Optical coherence tomography with a horizontal scan through the central fovea (lower Panel), reveals marked thickening and edema of the macula with cysts (C) and subretinal fluid (SRF). (Reproduced with permission from Antonetti, D. A., et al (2012). N Engl J Med 366(13):1227-1239, Copyright Massachusetts Medical Society)Streptozotocin-induced diabetic model
Various animal models have been developed for studying the molecular mechanisms underlying the pathogenesis of DR. Diabetes in animals is usually generated by pharmacological induction of hyperglycemia, feeding high-sugar diet, or spontaneously by selective inbreeding or genetic manipulation (50, 51). Because rodents show advantages of similar genetic background to humans, ease of handling and housing, relatively inexpensive and short reproductive cycles, they have been studied most extensively.
Streptozotocin (STZ) is an antibiotic produced by Streptomyces achromogenes, which can destroy pancreatic ß-cells which are responsible for endogenous insulin production. Thereby, STZ is widely used experimentally to induce insulin-dependent diabetes mellitus, also known as type 1 diabetes mellitus (52). STZ-induced hyperglycemia in rodents (predominantly, rat and mice) reproduces early symptoms of DR, such as thickening of the vascular basement membrane and leakage of iBRB (53, 54).
The need for isolated brain and retinal microvessels
To maintain the normal functions of brain and retina, the neural environment must be preserved within a narrow homeostatic range, which requires a tight regulation of exchange of nutrients, metabolites, ions, proteins and water between the blood and the neuronal tissue. As mentioned above, in mammals this is achieved within the microvessels of brain and retina (BMVs and RMVs, respectively) through their highly selective and protective barriers known as BBB and iBRB. In diabetes mellitus, both retinal and brain microvessels are directly exposed to a microenvironment with elevated concentration of glucose, so-called hyperglycemia. However, previous studies have demonstrated that the microvasculature in brain is not or obviously less susceptible to
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diabetes compared to the microvasculature in retina (55-57), where there is loss of the iBRB integrity as mentioned above. This might be surprising, as the retina is ontogenetically derived from the diencephalon, and its microvascular network shares, in many aspects, similar features with the cerebral microvascular network in terms of morphological and functional properties (58). These interesting observations produce a question: why the BMVs are able to keep “resistance” or are “less susceptible” to a diabetes insult, while the RMVs show high susceptibility to diabetes insult?Multiple methods have been used for comparing the structural, functional, and molecular profiles of the retinal and brain microvasculature. For instance, electron microscopy (59, 60) and digestion techniques (57, 61) were used to assess the morphometric features of brain and retinal microvascular. In fact, the value of any method largely depends on the measuring targets and how well this method supplies microvascular compartment with a high amount and specificity. Changes in gene expression often account for interactions between environmental stimuli and genetic materials, which underlie the regulation of various pathophysiological responses. Over the past decade, transcriptional analysis has become one of the most powerful approaches to assess gene expression and alterations under pathological conditions. To answer the above question, we attempted to measure the whole gene expression changes with microarray technology in retinal and brain microvasculature. Therefore, mechanically isolated microvessels are an important tool since they will avoid the inaccuracy induced by nonvascular tissues and prevent the degradation of RNA. In 1974, Brendel and his colleagues first isolated fresh bovine BMVs using a mechanical method of homogenization, centrifugation and filtration steps (62). Thereafter, using the same or modified protocols, scientists were able to extract BMVs from rat, mice, monkey and fish (63-67) as well as from human autopsy brains (68, 69). Using the isolated BMVs, a large number of transporters, receptors and tight junctions genes of the BBB were identified and quantified (70-73). Almost as early as BMVs, bovine RMVs were also successfully isolated using a modified mechanical method (74-76). However, this mechanical technique for bovine RMVs isolation has never been successfully transferred to rodents, probably because of too small starting-tissue (e.g. rat and mice retina) for isolating sufficient RMVs.
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Molecular profiles of the retinal and brain microvasculature
In order to uncover the molecular profiles of BMVs and RMVs in diabetic and nondiabetic conditions, we attempted to establish a new mechanical protocol that allowed isolation of high amounts and highly purified microvessels from the same rats. Thereafter, we aimed to first explore the gene expression properties of BMVs and RMVs in physiological conditions, and further aimed to determine whether BMVs and RMVs have different responses to hyperglycemia of diabetes at the gene expression level (Fig. 4).In chapter 3, we describe the development of a new mechanical isolation method for both RMVs and BMVs from healthy and diabetic rats, by which we were able to extract with high purity, high quality and intact RNA. Samples of full brain and retinal tissue were collected as controls, and we also harvested the pial arteries from the same rat. The purity of isolated microvessels was microscopically checked, followed by quantitative RT-PCR (qRT-PCR) to assess specific cell markers.
In chapter 4, using the newly established isolation method (described in chapter 3), we captured purified RMVs and BMVs samples from healthy rats. Gene expression differences were identified between RMVs and BMVs and these data provided clues towards deciphering the molecular mechanisms of organotypic vascular differentiation in healthy animals.
In chapter 5, also applying the isolation method described in chapter 3, we captured purified RMVs and BMVs samples from healthy and diabetic rats. Based on the global transcriptional analysis, we aimed to study whether BMVs had special protective factors to keep the brain microvasculature less susceptible to diabetes. In parallel, we also assessed the diabetic effects on gene expression profiles of RMVs, to demonstrate whether the balance of injury and protective factors in retinal microvasculature was affected by diabetes
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Figure 4. Overview of the brain and retinal microvessels (BMVs, and RMVs, respectively)
studies in this thesis. BT, brain tissue; RT, retinal tissue.
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67. Miller DS, Graeff C, Droulle L, Fricker S, & Fricker G (2002) Xenobiotic efflux pumps in isolated fish brain capillaries. American journal of physiology. Regulatory,
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of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy. Brain research 1134(1):1-11.72. Dauchy S, et al. (2008) ABC transporters, cytochromes P450 and their main transcription factors: expression at the human blood-brain barrier. Journal of
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73. Uchida Y, et al. (2011) Quantitative targeted absolute proteomics of human blood-brain barrier transporters and receptors. Journal of neurochemistry 117(2):333-345.
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76. Buzney SM, Frank RN, & Robison WG, Jr. (1975) Retinal capillaries: proliferation of mural cells in vitro. Science 190(4218):985-986.
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CHAPTER 2
Enhancement of bradykinin-induced relaxation by focal
brain ischemia in the rat middle cerebral artery: Receptor
expression upregulation and activation of multiple pathways
Youhai Lia,b, Natalia Lapinac, Nina Weinzierla, Lothar Schillinga
a
Division of Neurosurgical Research, Medical Faculty Mannheim, University of Heidelberg, Germany bCurrent address: Department of Pathology & Medical Biology, Medical Biology Section, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands c
Current address: Lytech Co. Ltd., Moscow, Russia
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Abstract
Purpose: Focal brain ischemia markedly affects cerebrovascular reactivity. So far,
these changes have mainly been related to alterations of smooth muscle cell function while alterations of the endothelial lining have not yet been studied in detail. We have, therefore, investigated the effects of ischemia/reperfusion injury on bradykinin (BK)-induced relaxation, since BK is an important mediator of tissue inflammation and affects vascular function in an endothelium-dependent manner.
Methods: Focal brain ischemia was induced in rats by endovascular filament occlusion
(2h) of the middle cerebral artery (MCA). After 22h reperfusion, both MCAs were harvested and the response to BK studied in organ bath experiments. Expression of the BK receptor subtypes 1 and 2 (B1, B2) was determined by real-time semi-quantitative RT-qPCR methodology, and whole mount immunofluorescence staining was performed to show the B2 receptor protein expression.
Results: In control animals, BK did not induce significant vasomotor effects despite a
functionally intact endothelium and robust expression of B2 mRNA. After ischemia/reperfusion injury, BK induced a concentration-related sustained relaxation in all arteries studied, more pronounced in the ipsilateral than in the contralateral MCA. The B2 mRNA was significantly upregulated and the B1 mRNA displayed de novo expression, again more pronounced ipsi- than contralaterally. Endothelial cells displaying B2 receptor immunofluorescence were observed scattered or clustered in previously occluded MCAs. Relaxation to BK was mediated by B2 receptor activation, abolished after endothelium denudation, and largely diminished by blocking nitric oxide (NO) release or soluble guanylyl cyclase activity. Relaxation to BK was partially inhibited by charybdotoxin (ChTx), but not apamin or iberiotoxin suggesting activation of an endothelium-dependent hyperpolarization pathway. When the NO-cGMP pathway was blocked, BK induced a transient relaxation which was suppressed by ChTx.
Conclusions: After ischemia/reperfusion injury BK elicits endothelium-dependent
relaxation which was not detectable in control MCAs. This gain of function is mediated by B2 receptor activation and involves the release of NO and activation of an
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endothelium-dependent hyperpolarization. It goes along with increased B2 mRNA and protein expression, leaving the functional role of the de novo B1 receptor expression still open.Introduction
The presence of kinins, as well as kinin-synthesizing and –destroying enzymes in the brain, was first described by Hori (1) in rabbits. Based on these and subsequent studies supporting and extending the initial findings, brain tissue is considered to express a full kallikrein-kinin system (KKS). The major biologically active component of the KKS, the nonapeptide bradykinin (BK), acts upon two receptor types termed subtype 1 (B1) and subtype 2 (B2) receptor. In the brain vasculature, BK exerts endothelium-dependent vasodilatation of arteries and arterioles and an increase in capillary permeability eventually leading to (enhancement of) vasogenic brain edema. In healthy conditions, these effects are mediated by activating the B2 receptor (2) which is constitutively expressed on the endothelial cells while the expression of the B1 receptor is typically below detection levels.
Despite much research performed in the past, the importance of the B1 and B2 receptors in vascular dysfunction and tissue damage following a traumatic or ischemic brain insult is still not fully understood. A major role of the B2 receptor may be derived from studies using selective receptor antagonists or B2 receptor knock-out mice showing a reduction of the volume of tissue, a decrease of brain edema, and improvement of neurological outcome in models of brain trauma (3-6) and ischemia (7-9). Moreover, in brain trauma patients, application of B2 receptor antagonists proved effective in counteracting the increase in intracranial pressure (10, 11). However, pharmacological inhibition or knock-out of the B1 receptor has also been shown to carry therapeutic benefit in a model of ischemic brain damage in mice (12). Thus, both B1, as well as B2 receptors, may be of functional importance in KKS activation and mediation of BK-induced effects in pathological situations.
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Alterations of cerebroarterial reactivity in pathological conditions accompanied by alterations of gene expression have been demonstrated for endothelin-1, angiotensin II, and 5-HT in models of focal brain ischemia (13-15). However, these studies have focused on the smooth muscle cells only, disregarding potential changes in endothelial cells, which play a pivotal role in the regulation of vascular tone and reactivity. We have, therefore, investigated the alterations of the vasomotor effects of BK following ischemia/reperfusion (I/R) injury in the rat middle cerebral artery (MCA). Bradykinin was chosen because (i) its vasomotor effects in cerebral arteries is endothelium-dependent, and (ii) activation of the KKS is a highly adaptive inflammatory response following ischemic and traumatic injury with a de novo expression ofthe B1 receptor appearing to be a hallmark (16). Although B1 receptor activation has been linked to vasomotor activity in peripheral arteries (17), no such data are available yet in cerebral arteries.Our results indicate a tremendous increase in the relaxation-inducing action of BK in rat MCA following I/R injury. Despite a de novo expression of the B1 receptor mRNA in the vessel wall, we found no indication of any vasomotor effect. In fact, relaxation mediated by B2 receptor activation was endothelium-dependent and involved release of nitric oxide (NO) and activation of an endothelium-dependent hyperpolarizing factor (EDHF)-related pathway. The enhanced vasomotor action went along with a significant upregulation of the B2 receptor on the level of gene expression and occurrence of a respective immunofluorescent signal in the endothelium. To the best of our knowledge, the present study is the first to describe I/R injury-related alterations of B1 and B2 receptors on the levels of gene expression, protein expression and vasomotor reactivity in the rat MCA.
Materials and methods
Animals
Male Sprague Dawley rats (body weight 300±500 g) from Janvier (Isle St. Genest, France) were used throughout. All animals were allowed to accommodate for at least one week before exper-iments were done. Approval of the experimental protocol was
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obtained from the Federal Ani-mal Ethics Committee (Regierungspraesidium Karlsruhe). The experiments were performed in compliance with the relevant laws and institutional guidelines for the care and use of animals in research according to the Directive 2010/63/EU. This includes all efforts to minimize pain and stress to the animals and limits to the number of animals used.Induction of focal ischemia and reperfusion
Focal brain ischemia was induced by the intravascular filament occlusion technique as described previously (18, 19). A surgical level of anesthesia was established with isoflurane (2.5% in a 70:30 air: O2 mixture) delivered through a face mask while spontaneously breathing. The skull was exposed and a hole created over the right hemisphere (1mm caudally / 4 mm occipitally from the bregma) leaving the inner bone layer intact. A glass fiber connected to a laser Doppler flowmetry (LDF) monitor (Moore DRT 4, Axminster, England) was attached to the hole for continuous recording of perfusion in the area supplied by the MCA. The animal was then turned into a supine position and the neck opened in the midline. The carotid bifurcation was identified on the right side and flow through the common carotid artery transiently blocked. The external carotid artery was ligated and mobilized. A nylon filament (4-0) coated with an elastomer (Provil novolight, Heraeus Kulzer, Hanau Germany; tip diameter, 430-460 μm) was introduced and advanced into the internal carotid artery until a sudden drop of the LDF signal indicated MCA occlusion. The filament was fixed to the external carotid artery, the block of the common carotid artery released and the LDF recording checked for signs of hemorrhage (20). Buprenorphine (3 μg / 100 g body weight) was injected subcutaneously to reduce postoperative pain, the glass fiber was removed, and after closure of all wounds the animal was returned to its cage to recover from anesthesia.
The animals were re-anesthetized with isoflurane 100 min after establishing MCA occlusion. The LDF-probe was re-inserted in the skull and the filament removed 2 h after positioning. A sharp increase of the LDF signal indicated successful reperfusion. Buprenorphine was again injected (half the dose given in the first surgical session) and after removal of the LDF fiber all wounds were closed again. Anesthesia was discontinued and the animal returned to its cage.
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Studies of vasoreactivity
At the end of the 24h observation period the animals were deeply anesthetized (5% isoflurane in oxygen) and sacrificed by bleeding from the carotid arteries. The skull was quickly opened and the brain carefully removed and transferred into a dish containing cold modified Krebs solution (composition in mM: NaCl, 119; KCl, 3.0; NaH2PO4, 1.2; CaCl2, 1.5; MgCl2, 1.2; NaHCO3, 15; glucose, 10). Both MCAs were meticulously isolated under a binocular microscope (GZ6, Zeiss, Oberkochem, Germany) and ring segments of approximately 2 mm length mounted onto 2 stainless steel wires (diameter, 30µm) for measurement of isometric force. In some experiments endothelium denudation was performed by rubbing the luminal surface of the segment with an eyelash as described previously (21).
After mounting, all segments received a 90 min accommodation period with warming of the bath solution to 37°C and repeated correction of the resting tension until a stable level of approximately 2.5 mN was reached. Tension was measured by force transducers (F10, coupled to TAM-A amplifiers, both from Hugo Sachs, March-Hugstetten, Germany). The signals from the force detection systems were fed into a computer and continuously recorded using a purpose-built program based on LabView (Munich, Germany) as described previously (22).
After accommodation all segments underwent standard tests to prove (i) the functional integrity of the contractile apparatus by immersion in Krebs solutions containing increasing concentrations of K+ (50, 80, and 124 mM, NaCl substituted by KCl in equimolar amounts) and (ii) the presence or successful removal of the endothelium by cumulative application of sarafotoxin 6c (10-8 and 10-7 M) following precontraction with U46619 (3x10-7 M). Sarafotoxin 6c selectively activates the endothelin B receptor subtype located in endothelial cells resulting in release of NO (23).
The vasomotor effect of BK was studied after precontraction with U46619 (we never observed contraction when BK was applied upon resting tension). Receptor characterization was done by incubating ring segments for at least 30 minutes with the selective B1 receptor antagonist lys-(des-arg9, leu8)-BK ((10-5 M) or the selective B2 receptor antagonist Hoe140 (10 -6 M) before application of BK. In addition, we also tested the effect of the selective B1 receptor agonist lys-(des-arg9)-BK (10-12 - 10-6 M) in precontracted segments. In order to characterize the mechanisms involved in
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mediating BK-induced relaxation after I/R injury, we used blockers of NO release and inhibitors of Ca2+-dependent K+ (KCa) channels. In each case, the segments were exposed to the blockers for at least 30 minutes before precontraction with U46619 and application of BK.
Studies of gene expression
For studies of gene expression individual MCAs were used. The arteries were carefully cleaned from all adhering tissue, transferred into cell lysis buffer (RLT buffer [Qiagen, Hilden, Germany], 350 µl plus 3.5 µl mercaptoethanol), and immediately cooled down to -75°C until further use. Each MCA underwent mechanical disruption using a motor-driven homogenizer (Homgen, Schuett Biotec, Goettingen, Germany), shearing of DNA by repeated pushing through a 30G needle, and extraction of total RNA using a commercially available kit (RNeasyplus, Qiagen). Concentration and integrity of the total RNA obtained were measured using Picochips read in a 2100 Bioanalyzer (both from Agilent Technologies, Waldbronn, Germany).
For reverse transcription 10µl total RNA solution obtained from individual arteries was mixed with 1 µl random hexamers (200 ng/µl) and 1 µl oligo (dT)18 solution (10 µM; both from Sigma-Aldrich), heated to 70° C for 5 min and immediately cooled down on ice. Thereafter, 8 µl master mix (4 µl 5x RT-buffer; 2 µl dNTP mix [5 mM each], 1 µl RNase Inhibitor [RNasin® Plus, 20 U/µl]; all from Promega), 1 µl Sensiscript® reverse transcriptase [200 U/µl]; Qiagen) were added and the solution incubated at 37° C for 90 min (PCR Express, Hybaid Thermo Scientific). The reaction was stopped by increasing to 94° C for 10 min.
Gene expression was measured by real-time semi-quantitative real-time PCR (RT-qPCR) methodology using 1 µl of cDNA solution which was mixed with 8 µl PCR-grade H2O, 10 µl Absolute™QPCR Mix (Thermo Scientific, Hamburg, Germany), and 1 µl of a probe specific primer mixture (TaqMan® Gene expression Assay [20x], Applied Biosystems). We used commercially available hydrolysis probes (TaqMan®, Applied Biosystems, Darmstadt, Germany). The assay designations (in brackets are given the gene name, the accession number, the respective assay number, the assay location, and the amplicon length) are elongation factor 1 (EeF1a2, NM_012660.2, #Rn00561973_m1, 1592, 82bp), B1 receptor (Bdkrb1, NM_030851.1,
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#Rn00578261_m1, 86; 105 bp), and B2 receptor (Bdkrb2, NM_173100.2, #Rn00597384_m1, 209, 75bp). The amplification protocol consisted of an initial heating period (95 °C for 15 min) followed by 40 amplification cycles (95 °C for 15 sec and 60 °C for 1 min). Experiments were performed using either a Stratagene MxPro 3005P light cycler (Stratagene Europe, Amsterdam, The Netherlands) or a StepOne light cycler (Applied Biosystems). In preliminary studies we confirmed that both systems yielded fully comparable results. Measurements were done at least in duplicates and the cycle threshold (CT) values averaged to be used for further analyses.Immunofluorescence detection of B2 receptor protein expression
Whole mount immunofluorescence microscopy was performed to detect B2 receptor protein expression in the MCA wall. Arteries were cut along their long axis and immersed in 4% paraformaldehyde for 10 minutes at room temperature. Subsequently, free-floating staining was performed in 96 well plates at room temperature unless otherwise stated. The protocol with washes between the incubation steps consisted of (i) incubation in TritonX100 (1% dissolved in saline solution PBS for 30 minutes), (ii) immersion in serum free protein block (DAKO X0909, DAKO, Hamburg, Germany) for 60 minutes, (iii) incubation with a primary antibody raised against the B2 receptor protein (mouse monoclonal α B2R, sc-136216, Santa Cruz, Heidelberg, Germany; dilution, 1:300 overnight at 4°C), (iv) incubation with the secondary antiserum (goat anti mouse IgG, Invitrogen A11004 Alexa Fluor 568, Thermo Fisher Scientific, Darmstadt, Germany; dilution 1:1000 for 3 h), (v) nuclear counterstaining with 4′,6-Diamidin-2-phenylindol (DAPI, 300 nM), (vi) covering (Roti®-Mount FluorCare HP19.1, Carl Roth, Karlsruhe, Germany). The segments were examined under a fluorescence microscope (Z1) equipped with an Axiocam camera (both from Carl Zeiss, Jena, Germany).
Measurement of ischemic brain damage
The volume of ischemic damage was determined based on the high contrast silver nitrate staining method introduced by Vogel and coworkers (24). Briefly, serial cryosections of the brain (thickness, 20 µm; distance 1 mm) were air-dried, immersed in an impregnation solution for 2 min (composition: 5ml of a 10% silver nitrate solution
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and 10ml saturated lithium carbonate solution were mixed and after dissolving the white precipitate by adding 25% ammonia drop by drop 75ml distilled water were added), washed in distilled water (6 x 1 min) and subsequently immersed in a developing solution for 3 min (0.3 g hydroquinone, 15 ml acetone and 1.1g trisodium citrate dissolved in 20ml formaldehyde (37%) / 70ml distilled water), washed again before covering with Eukitt medium (O. Kindler GmbH, Bobingen, Germany). Volumetric analysis of the brain damage was performed as described in detail previously (19).Compounds
The chemicals used in the present study along with the suppliers are BK acetate salt, lys-(des-arg9)-BK trifluoroacetate salt (B1 receptor agonist), lys-(des-arg9,leu8)-BK (B1 receptor antagonist) and iberiotoxin from Bachem (Heidelberg, Germany), Hoe 140 (Arg-Arg-Pro-Hyp-Gly-Thi-Ser-Tic-Oic-Arg, B2 receptor antagonist), charybdotoxin (ChTx), Nω–nitro-l-arginine (L-NNA), trisodium citrate dihydrate, hydroquinone, and carbolithium from Sigma-Aldrich (Taufkirchen, Germany); sarafotoxin 6c, U46619 (9,11-Dideoxy-9a,11a-methanoepoxy prostaglandin F2a) and ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) from Enzo Life Science (Lausanne, Switzerland); 7-nitroindazole (7-NI) sodium salt from Santa Cruz Biotechnology (Heidelberg, Germany); apamin from RBI (Köln, Germany); NaCl, KCl, MgCl2, NaHCO3, glucose, and 25% ammonia solution from ROTH (Karlsruhe, Germany); NaH2PO4, CaCl2, silver nitrate, formaldehyde and acetone from Merck (Darmstadt, Germany); buprenorphine hydrochloride (Temgesic™; Essex Pharma, Munich, Germany); isoflurane (Forene™, AbbVie, Ludwigshafen, Germany).
Calculations and statistical analyses
In myograph studies relaxation is expressed in percent of precontraction induced by U46619 (300 nM). Levels of gene expression in the MCA wall were determined by means of the ΔCT method with elongation factor-1 serving as house-keeping gene. Ischemia-induced alterations of gene expression was determined using the comparative threshold cycle method (ΔΔCT methodology) and subsequently accumulation or depletion was calculated as fold change with
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The SigmaPlot software package (vers. 12.5; Systat Software GmbH, Erkrath, Germany) was employed for statistical analysis and preparation of the graphs. Statistical analysis was done using unpaired Student's t-test or if appropriate ANOVA procedure along with the Tukey test for post-hoc pairwise comparisons. All values are given as mean ± SEM along with the numbers of observations. In addition, fold changes are given along with the 95% confidence intervals.Results
A total of 42 rats underwent MCA occlusion. Among these, 5 were excluded because of a premature death. Occlusion of the MCA origin resulted in development of focal ischemic damage throughout all animals included in the analysis. Hemispheric swelling was 36 ± 2.5% indicating the presence of marked vasogenic edema. The volume of ischemic damage was 340 ± 16 mm³ (n= 28) after appropriate correction for brain swelling.
Studies in MCAs from control animals
In cerebral arteries obtained from control rats BK did not induce any significant vasomotor effect, neither under resting tension conditions nor following precontraction with U46619 (3x10-7 M). However, in each of these segments sarafotoxin 6c induced significant relaxation (maximum effect, 57 ± 5.2%; n= 25) indicating that the failure of BK to elicit relaxation cannot be ascribed to (functional) damage of the endothelium. Despite the lack of a significant vasomotor effect we found the B2 message present in all MCA vessels studied by means of RT- qPCR methodology (see Table 1).
Marked relaxation by BK occurring after transient MCA occlusion goes along with an upregulation of B1 and B2 receptor gene expression
After 2h of focal ischemia / 22h of recirculation BK induced a pronounced concentration-related relaxation which was well sustained in all MCA ring segments studied. This was true in the previously occluded MCA, and it was also true in the contralateral arteries, although at a significantly lower degree (Fig. 1). A stable level of
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precontraction induced by U46619 (3x10-7 M) was found in time-matched solvent control experiments indicating that the relaxation was not due to spontaneous loss of tension. Furthermore, relaxation was fully reproducible when BK was applied repeatedly (Fig. 1). Relaxation upon BK also occurred in segments precontracted by immersion in 50 mM K+ Krebs solution, although the maximum value observed (46 ± 8.5%, n= 8) was considerably smaller than in U46619-precontracted segments. The relaxation-inducing effect of BK went along with a significant upregulation of the B2 mRNA expression which was more pronounced in the MCAs from the ischemic hemisphere than in the contralateral arteries. In addition, there was a de novo expression of the B1 mRNA in the MCA vessel wall, again somewhat higher in the arteries taken from the right (ischemic) than from the left (contralateral) hemisphere (Table. 1). These changes in gene expression were highly robust since they were found in all vessels studied.Figure 1. Concentration-related relaxation induced by bradykinin (BK) of middle
cerebral artery (MCA) ring segments precontracted with U46619 (3x10-7 M). The ring
segments were obtained from rats which had undergone a 2h MCA occlusion followed by 22h of reperfusion. Shown are the results for the right (ipsilateral) and left (contralateral) MCA. The vasomotor effects were comparable upon the first and second application of BK in both sides.
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Solvent indicates time-matched solvent control measurements to check for the stability of precontraction. Given are mean ± SEM based on ≥ 7 observations.Table 1. Expression levels of the bradykinin subtype 1receptor (B1) and subtype 2 receptor (B2) mRNAin rat middle cerebral arteries (MCA) obtained under control conditions and after filament occlusion of the MCA origin (2h occlusion followed by 22h of reperfusion).
Control 24 h after transient MCA occlusion
MCA ipsilateral MCA contralateral
ΔCT ΔCT Fold upregulation ΔCT Fold upregulation B1 mRNA n.d. 0.88±0.42 de novo expression 1.30±0.39 de novo expression B2 mRNA 2.7±0.4 -2.44±0.59** x36 (16–81) 0.56±1.21* x9.8 (2–51) Indicated are the ΔCT values using elongation factor (EF)-1 as house-keeping gene as well as
the fold up-regulation based upon the ΔΔCT methodology. Given are mean ± SEM (≥4 samples)
while the average fold change is indicated along with the 95% confidence intervals in brackets. n.d., not detectable. *p<0.05 vs. control, **p<0.01 vs. control MCA.
BK-induced relaxation after transient MCA occlusion is mediated by B2 receptor activation: functional and immunofluorescence evidence
In order to characterize the receptor subtype(s) underlying BK-induced relaxation following I/R injury, specific agonists and antagonist were used. In the ipsilateral MCA, the B1 agonist lys-(des-arg⁹)-BK up to 1µM was devoid of any vasomotor effect, both under resting tension conditions as well as in precontracted segments. Furthermore, BK-induced relaxation was completely unaffected in the presence of the B1 antagonist lys-(des-arg⁹, leu⁸)-Bk (10-5 M). In contrast, the selective B2 antagonist Hoe140 (10-6 M) abrogated the vasomotor effect of BK suggesting B2 activation to solely account for the relaxation induced by BK following I/R injury (Fig. 2). Comparable effects were observed in ring segments from the contralateral MCA (Fig. S1).
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Figure 2. Effects of selective bradykinin (BK) receptor antagonists on the relaxation induced by bradykinin in rat middle cerebral artery (MCA) ring segments precontractedwith U46619 (3x10-7 M). Segments were obtained 24h following transient MCA occlusion
from the ischemic side. The BK-induced relaxation was not at all affected by the selective B1
receptor (Bk1R) antagonist lys-(des-arg⁹, leu⁸)-BK (10-5 M) while it was completely abrogated
in the presence of the selective B2 receptor (Bk2R) antagonist Hoe140 (10-6 M). Indicated are
mean ± SEM based on ≥ 6 observations. *p<0.05, ***p<0.001 vs. BK alone.
Whole mount immunofluorescence staining to detect B2 receptor protein revealed cells displaying high fluorescent intensity either scattered or arranged in a cluster-like fashion in displaying a low or even undetectable intensity level. This is exemplified in an image shown in figure 3. There was no signal detectable in smooth muscle cells in any artery studied. Control experiments with omission of the primary antibody did not show staining of the vessel wall. Furthermore, we never observed any significant immunofluorescence in MCAs taken from non-occluded animals.
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Figure 3. Whole mount immunofluorescence staining for the expression of B2 receptors in the middle cerebral artery wall following ischemia/reperfusion injury. Structures in blueare nuclei, while the B2 immunofluorescence presents in green. The endothelial cell nuclei run in the length axis of the artery while the smooth muscle cell nuclei are arranged perpendicular to the endothelial cells. Filled arrows mark endothelial cells displaying high level of immunofluorescence while the empty arrow points to an endothelial cell with low level staining. Asterisks mark smooth muscle cell nuclei (randomized choice) which are not in the focus of the image due to the thickness of the vessel wall. The clustering of endothelial cells displaying a high level of B2 receptor immunofluorescence was a typical finding, and we never found any immunofluorescent signal in smooth muscle cells.
BK-induced relaxation after transient MCA occlusion is mediated by release of NO and activation of an EDHF-related pathway
We studied the role of NO in mediating BK-induced relaxation after transient MCA occlusion using 7-NI (10-6 M), a selective neuronal NOS (nNOS) inhibitor, 1400W (a selective inhibitor of the inducible NOS (iNOS), 10-6 M), and Nω–nitro-l-arginine (L-NNA, 10-6 M - 10-5 M, an unselective NOS inhibitor). While 7-NI and 1400W did not significantly affect the BK-induced response (Fig. S2), L-NNA inhibited relaxation in a concentration-related manner as shown in figure 4. Furthermore, ODQ, a selective inhibitor of the soluble guanylyl cyclase completely blocked BK-induced relaxation
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(Fig. 4). Moreover, L-NNA at 10-5 M and ODQ (10-6 M) both increased the basal contractile force of the segments indicating involvement of the NO-cGMP pathway in adjusting resting tension. These results suggest that NO released from the endothelial cells is of major functional importance in the control of wall tension and mediation of BK-induced relaxation. In accord, we found this response abrogated in endothelium-denuded segments.Figure 4. Role of the NO-cGMP axis in bradykinin-induced relaxation in rat middle cerebral arteries obtained 24 h after induction of focal brain ischemia. Preincubation with
Nω-nitro-l-arginine (L-NNA) inhibited BK-induced relaxation in a concentration-related manner. Similarly, inhibition of the soluble guanylyl cyclase by ODQ abrogated the effect of BK. Indicated are mean ± SEM based on ≥ 4 observations. *p<0.05, **p<0.01 vs control.
Although NO released from endothelial cells was identified to play a major role in mediating BK-induced relaxation following transient MCA occlusion, there is evidence in favor of a membrane hyperpolarization-induced mechanism to be involved as well. This evidence is based on several observations including (i) a marked decrease of the maximal relaxation achieved with BK following precontraction in a 50 mM K+ Krebs solution as mentioned above, and (ii) the occurrence of a transient relaxant response in
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the presence of 10-5 M L-NNA (but not with 10-6 M) as shown in figure 5 and similarly in the presence of ODQ (10-6 M).Figure 5. In the presence of Nω–nitro-l-arginine (L-NNA, 10-5 M) bradykinin (BK) elicits
a transient relaxation in middle cerebral artery segments after transient focal brain ischemia. (A) Representative trace from a segment which was precontracted with U46619
(3x10-7 M). Application of high concentrations of BK (10-7 M to 10-5 M) induced transient
peaks of relaxation. (B) Mean values of the transient peaks of relaxation induced by high concentrations of BK in segments obtained from the previously occluded middle cerebral artery. Indicated are mean ± SEM (n=5).
In order to elucidate the mechanism involved in the generation of the transient relaxant response, we preincubated segments with different inhibitors of KCa channels before precontraction with U46619 and administration of BK. The results shown in figure 6 indicate that ChTx decreased the relaxant response to high concentrations of BK while neither apamin nor iberiotoxin significantly affected BK-induced relaxation. In addition, ChTx increased the inhibitory effect of L-NNA (10-6 and 10-5 M each) on the sustained phase of the BK-induced response, but this effect did not reach statistical significance. Moreover, the transient relaxation upon BK occurring in MCA segments with the NO-cGMP axis inhibited (see Fig. 5) was completely suppressed by ChTx. Based on the spectrum of actions with ChTx blocking large and intermediate conductance channels and iberiotoxin and apamin acting as selective blockers of large and small conductance channels (26) these results strongly suggest the involvement of intermediate conductance KCa channels underlying the EDHF-related pathway activated by BK.
