University of Groningen Brain and retinal macro- and microvasculature Li, Youhai

Brain and retinal macro- and microvasculature Li, Youhai

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Li, Y. (2018). Brain and retinal macro- and microvasculature: Response to ischemic and hyperglycemic stress. University of Groningen.

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CHAPTER 1

General Introduction

CHAPTER 1

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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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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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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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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.

References

1. Karbowski J (2011) Scaling of brain metabolism and blood flow in relation to capillary and neural scaling. PloS one 6(10):e26709.

2. Wong AD, et al. (2013) The blood-brain barrier: an engineering perspective. Frontiers

in neuroengineering 6:7.

3. Brinker T, Stopa E, Morrison J, & Klinge P (2014) A new look at cerebrospinal fluid circulation. Fluids and barriers of the CNS 11:10.

4. Pries AR & Secomb TW (2014) Making microvascular networks work: angiogenesis, remodeling, and pruning. Physiology 29(6):446-455.

5. Serlin Y, Shelef I, Knyazer B, & Friedman A (2015) Anatomy and physiology of the blood-brain barrier. Seminars in cell & developmental biology 38:2-6.

6. Chow BW & Gu C (2015) The molecular constituents of the blood-brain barrier. Trends

I

澳

21

澳 澳

7. Jacobson M & Hirose G (1978) Origin of the retina from both sides of the embryonic brain: a contribution to the problem of crossing at the optic chiasma. Science 202(4368):637-639.

8. London A, Benhar I, & Schwartz M (2013) The retina as a window to the brain-from eye research to CNS disorders. Nature reviews. Neurology 9(1):44-53.

9. Nguyen CTO, et al. (2017) Retinal biomarkers provide "insight" into cortical pharmacology and disease. Pharmacology & therapeutics 175:151-177.

10. Harada T, Harada C, & Parada LF (2007) Molecular regulation of visual system development: more than meets the eye. Genes & development 21(4):367-378.

11. Hayreh SS (2011) Acute retinal arterial occlusive disorders. Progress in retinal and eye

research 30(5):359-394.

12. Campbell M & Humphries P (2012) The blood-retina barrier: tight junctions and barrier modulation. Advances in experimental medicine and biology 763:70-84.

13. Faraci FM & Heistad DD (1990) Regulation of large cerebral arteries and cerebral microvascular pressure. Circulation research 66(1):8-17.

14. Obermeier B, Daneman R, & Ransohoff RM (2013) Development, maintenance and disruption of the blood-brain barrier. Nature medicine 19(12):1584-1596.

15. Furchgott RF & Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288(5789):373-376. 16. Ozkor MA & Quyyumi AA (2011) Endothelium-derived hyperpolarizing factor and

vascular function. Cardiology research and practice 2011:156146.

17. Garland CJ, Hiley CR, & Dora KA (2011) EDHF: spreading the influence of the endothelium. British journal of pharmacology 164(3):839-852.

18. Hein TW, Rosa RH, Jr., Yuan Z, Roberts E, & Kuo L (2010) Divergent roles of nitric oxide and rho kinase in vasomotor regulation of human retinal arterioles. Investigative

ophthalmology & visual science 51(3):1583-1590.

19. Faraci FM & Heistad DD (1998) Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiological reviews 78(1):53-97.

20. Triggle CR, et al. (2012) The endothelium: influencing vascular smooth muscle in many ways. Canadian journal of physiology and pharmacology 90(6):713-738. 21. Kunz A & Iadecola C (2009) Cerebral vascular dysregulation in the ischemic brain.

Handbook of clinical neurology 92:283-305.

22. Winters A, et al. (2012) Transient focal cerebral ischemia induces long-term cerebral vasculature dysfunction in a rodent experimental stroke model. Translational stroke

research 3(2):279-285.

23. Palomares SM & Cipolla MJ (2014) Myogenic tone as a therapeutic target for ischemic stroke. Current vascular pharmacology 12(6):788-800.

I

澳

24. Yildiz O, Seyrek M, & Gul H (2013) Pharmacology of Arterial Grafts for Coronary

Artery Bypass Surgery.

25. Ehrlich P (1885) Das Sauerstoff-Bedürfniss des Organismus: eine farbenanalytische studie. Berlin, Hirschwald.

26. Lewandowsky M (1900) Zur lehre vonder cerebrospinalflussigkeit. Z. Klin. Med. 40:480-494.

27. Palm E (1947) On the occurrence in the retina of conditions corresponding to the blood-brain barrier. Acta ophthalmologica 25(1):29-35.

28. Bradbury MW (1993) The blood-brain barrier. Experimental physiology 78(4):453-472. 29. Ballabh P, Braun A, & Nedergaard M (2004) The blood-brain barrier: an overview:

structure, regulation, and clinical implications. Neurobiology of disease 16(1):1-13. 30. Butt AM, Jones HC, & Abbott NJ (1990) Electrical resistance across the blood-brain

barrier in anaesthetized rats: a developmental study. The Journal of physiology 429:47-62.

31. Huber JD, Egleton RD, & Davis TP (2001) Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier. Trends in neurosciences 24(12):719-725. 32. Pournaras CJ, Rungger-Brandle E, Riva CE, Hardarson SH, & Stefansson E (2008)

Regulation of retinal blood flow in health and disease. Progress in retinal and eye

research 27(3):284-330.

33. Hirschi KK & D'Amore PA (1996) Pericytes in the microvasculature. Cardiovascular

research 32(4):687-698.

34. Beltramo E & Porta M (2013) Pericyte loss in diabetic retinopathy: mechanisms and consequences. Current medicinal chemistry 20(26):3218-3225.

35. Daneman R, Zhou L, Kebede AA, & Barres BA (2010) Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature 468(7323):562-566.

36. Trost A, et al. (2016) Brain and Retinal Pericytes: Origin, Function and Role. Frontiers

in cellular neuroscience 10:20.

37. Zheng Y, Ley SH, & Hu FB (2018) Global aetiology and epidemiology of type 2 diabetes mellitus and its complications. Nature reviews. Endocrinology 14(2):88-98. 38. Kitada M, Zhang Z, Mima A, & King GL (2010) Molecular mechanisms of diabetic

vascular complications. Journal of diabetes investigation 1(3):77-89.

39. Suryavanshi SV & Kulkarni YA (2017) NF-kappabeta: A Potential Target in the Management of Vascular Complications of Diabetes. Frontiers in pharmacology 8:798. 40. Barrett EJ, et al. (2017) Diabetic Microvascular Disease: An Endocrine Society Scientific Statement. The Journal of clinical endocrinology and metabolism 102(12):4343-4410.

I

澳

23

澳 澳

injury and protective factors. Cell metabolism 17(1):20-33.

42. Lee R, Wong TY, & Sabanayagam C (2015) Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss. Eye and vision 2:17.

43. Cheung N, Mitchell P, & Wong TY (2010) Diabetic retinopathy. Lancet 376(9735):124-136.

44. Klaassen I, Van Noorden CJ, & Schlingemann RO (2013) Molecular basis of the inner blood-retinal barrier and its breakdown in diabetic macular edema and other pathological conditions. Progress in retinal and eye research 34:19-48.

45. Cunha-Vaz J, Faria de Abreu JR, & Campos AJ (1975) Early breakdown of the blood-retinal barrier in diabetes. The British journal of ophthalmology 59(11):649-656. 46. Duh EJ, Sun JK, & Stitt AW (2017) Diabetic retinopathy: current understanding,

mechanisms, and treatment strategies. JCI insight 2(14).

47. Ehrlich R, et al. (2010) Diabetic macular oedema: physical, physiological and molecular factors contribute to this pathological process. Acta Ophthalmol 88(3):279-291.

48. Antonetti DA, Klein R, & Gardner TW (2012) Diabetic retinopathy. The New England

journal of medicine 366(13):1227-1239.

49. Das A (2016) Diabetic Retinopathy: Battling the Global Epidemic. Investigative

ophthalmology & visual science 57(15):6669-6682.

50. Lai AK & Lo AC (2013) Animal models of diabetic retinopathy: summary and comparison. Journal of diabetes research 2013:106594.

51. Rees DA & Alcolado JC (2005) Animal models of diabetes mellitus. Diabetic medicine :

a journal of the British Diabetic Association 22(4):359-370.

52. Szkudelski T (2001) The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiological research 50(6):537-546.

53. Robinson R, Barathi VA, Chaurasia SS, Wong TY, & Kern TS (2012) Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals. Disease models & mechanisms 5(4):444-456.

54. Do carmo A, Ramos P, Reis A, Proenca R, & Cunha-vaz JG (1998) Breakdown of the inner and outer blood retinal barrier in streptozotocin-induced diabetes. Experimental

eye research 67(5):569-575.

55. Badr GA, Tang J, Ismail-Beigi F, & Kern TS (2000) Diabetes downregulates GLUT1 expression in the retina and its microvessels but not in the cerebral cortex or its microvessels. Diabetes 49(6):1016-1021.

56. Dai J, Vrensen GF, & Schlingemann RO (2002) Blood-brain barrier integrity is unaltered in human brain cortex with diabetes mellitus. Brain research 954(2):311-316. 57. Kern TS & Engerman RL (1996) Capillary lesions develop in retina rather than cerebral

I

澳

cortex in diabetes and experimental galactosemia. Archives of ophthalmology 114(3):306-310.

58. Patton N, et al. (2005) Retinal vascular image analysis as a potential screening tool for cerebrovascular disease: a rationale based on homology between cerebral and retinal microvasculatures. Journal of anatomy 206(4):319-348.

59. Frank RN, Dutta S, & Mancini MA (1987) Pericyte coverage is greater in the retinal than in the cerebral capillaries of the rat. Investigative ophthalmology & visual science 28(7):1086-1091.

60. Frank RN, Turczyn TJ, & Das A (1990) Pericyte coverage of retinal and cerebral capillaries. Investigative ophthalmology & visual science 31(6):999-1007.

61. Cogan DG & Kuwabara T (1984) Comparison of retinal and cerebral vasculature in trypsin digest preparations. The British journal of ophthalmology 68(1):10-12.

62. Brendel K, Meezan E, & Carlson EC (1974) Isolated brain microvessels: a purified, metabolically active preparation from bovine cerebral cortex. Science 185(4155):953-955.

63. Goldstein GW, Wolinsky JS, Csejtey J, & Diamond I (1975) Isolation of metabolically active capillaries from rat brain. Journal of neurochemistry 25(5):715-717.

64. Dogrukol-Ak D, et al. (2009) Isolation of peptide transport system-6 from brain endothelial cells: therapeutic effects with antisense inhibition in Alzheimer and stroke models. Journal of cerebral blood flow and metabolism : official journal of the

International Society of Cerebral Blood Flow and Metabolism 29(2):411-422.

65. Betz AL, Csejtey J, & Goldstein GW (1979) Hexose transport and phosphorylation by capillaries isolated from rat brain. The American journal of physiology 236(1):C96-102. 66. Ito K, et al. (2011) Quantitative membrane protein expression at the blood-brain barrier

of adult and younger cynomolgus monkeys. Journal of pharmaceutical sciences 100(9):3939-3950.

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,

integrative and comparative physiology 282(1):R191-198.

68. Andjelkovic AV, Spencer DD, & Pachter JS (1999) Visualization of chemokine binding sites on human brain microvessels. The Journal of cell biology 145(2):403-412. 69. Grammas P (2000) A damaged microcirculation contributes to neuronal cell death in

Alzheimer's disease. Neurobiology of aging 21(2):199-205.

70. Enerson BE & Drewes LR (2006) The rat blood-brain barrier transcriptome. Journal

of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 26(7):959-973.

I

澳

25

澳 澳

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

neurochemistry 107(6):1518-1528.

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.

74. Meezan E, Brendel K, & Carlson EC (1974) Isolation of a purified preparation of metabolically active retinal blood vessels. Nature 251(5470):65-67.

75. Hjelle JT, Baird-Lambert J, Cardinale G, Specor S, & Udenfriend S (1978) Isolated microvessels: the blood-brain barrier in vitro. Proceedings of the National Academy of

Sciences of the United States of America 75(9):4544-4548.

76. Buzney SM, Frank RN, & Robison WG, Jr. (1975) Retinal capillaries: proliferation of mural cells in vitro. Science 190(4218):985-986.