1

Chronic hypertension increases susceptibility to acute

IOP challenge in rats

Zheng He1, Algis J Vingrys1, James A Armitage2,3, Christine TO Nguyen1, Bang V Bui1

1. Department of Optometry & Vision Sciences, University of Melbourne, Parkville,

3010, Victoria, Australia.

2. School of Medicine (Optometry), Deakin University, Waurn Ponds, Victoria, 3216,

Australia

3. Department of Anatomy and Developmental Biology, Monash University, Clayton

3800, Victoria, Australia

Corresponding author: Dr. Bang V Bui, Department of Optometry & Vision Sciences,

University of Melbourne, Parkville, 3010, Victoria, Australia.

Ph: +61 3 9349 7521

Fx: +61 3 9349 7498

email: bvb@unimelb.edu.au

Running title: chronic high blood pressure and IOP challenge

Key words: intraocular pressure, chronic high blood pressure, electroretinogram,

retinal blood flow

Word count: total, 4070; abstract, 243; introduction, 427; methods, 1649; results,

629; discussion, 1122

Number of Figures: 4

Number of Tables: 0

2

Abstract 1

Purpose: to consider the effect of chronic arterial hypertension on the susceptibility of 2

the retina to acute intraocular (IOP) challenge. 3

Methods: Anesthetized adult Long-Evans rats with normal (n=5, receiving saline 4

subcutaneously), chronic high blood pressure (BP) for 4 weeks (n=15, Angiotensin II 5

subcutaneously) and acute high BP for 1 hour (n=10, Angiotensin II intravenously) 6

underwent IOP elevation (10-120 mmHg, 5 mmHg steps each 3 min). During IOP 7

elevation, retinal function and ocular blood flow were monitored with electroretinogram 8

(ERG) and laser-Doppler flowmetry (LDF) respectively. BP was monitored via a femoral 9

artery cannula. ERG and LDF responses are expressed as a percentage of baseline and 10

compared between groups. The left ventricle and the aorta were dissected to assess the 11

morphological changes associated with chronic hypertension. 12

Results: 4-weeks of hypertension (systolic BP 192 ± 4 mmHg) produced cardiac 13

hypertrophy and thickened aortic arterial walls compared with controls (systolic BP 112 ± 14

3 mmHg). Retinal function was unaltered with chronic hypertension compared with 15

normotensive animals. During acute IOP elevation, ERG and LDF were reduced in a 16

dose-dependent manner in all BP groups. Both chronic and acute hypertension made 17

the ERG and LDF less susceptible to IOP elevation. However, the degree of resistance 18

to IOP elevation was greater in acute hypertension compared with chronic hypertension 19

(p<0.05). 20

Conclusion: Acute blood pressure elevation makes retinal function and blood flow less 21

susceptible to IOP elevation. The reduced susceptibility afforded by improved ocular 22

perfusion pressure is compromised after 4-weeks of chronic hypertension. 23

24

3

Introduction 25

Ocular perfusion pressure represents the balance between the opposing forces of blood 26

pressure and intraocular pressure (OPP = BP – IOP). Studies have shown that 27

short-term blood pressure elevation increases the IOP threshold needed to induce retinal 28

dysfunction and blood flow attenuation.1-4 Whether this is also the case in chronic arterial 29

hypertension remains unclear. 30

Chronic hypertension is commonly associated with structural changes in the 31

cardiovascular system such as cardiac muscle remodeling and arthrosclerosis. These 32

changes reduce cardiac output and increase the resistance in peripheral arteries, 33

therefore leading to greater risk of ischemic heart disease and stroke, which is contrary 34

to the expectation that better organ perfusion pressure should be beneficial. In the eye, 35

whether chronic hypertension affords protection against, or increases the vulnerability to 36

IOP elevation has clinical relevance for glaucoma. As evidenced by the end organ 37

damage to the retina, kidney, heart and brain that can occur in chronic hypertension, it 38

seems that higher blood pressure does not always equate to better perfusion pressure. 39

Studies have shown that low BP, including greater nocturnal BP dipping5, 6 and overly 40

aggressive treatment of systemic hypertension,7, 8 is associated with a greater risk of 41

glaucoma development. These outcomes are consistent with beneficial effects of a high 42

OPP. A review of epidemiological studies found conflicting outcomes regarding 43

associations between systemic hypertension and glaucoma.9 The Baltimore Eye Survey 44

found that systemic hypertension appears to be protective against glaucoma in younger 45

patients but a risk factor in older patients.10 Therefore the authors proposed that in early 46

hypertension, prior to blood vessel compromise secondary to atherosclerosis, patients 47

are likely to benefit from improved OPP and blood flow to the eye. This protective effect 48

may become negated later in the course of disease when vascular damage becomes 49

more dominant.10, 11 One way to test this hypothesis would be to compare how the retina 50

4

copes with IOP elevation the effect of acute and chronic hypertension on OPP 51

modification. As yet there is no compelling experimental evidence that chronic 52

hypertension is less protective than is an equivalent acute hypertensive increase in OPP, 53

despite the presence of atherosclerotic changes which are known to be associated with 54

the chronic state. 55

In this study, we compared the effect of acute (1-hour) and chronic (4-week) BP 56

elevation on the susceptibility of the ERG and ocular blood flow in response to acute IOP 57

elevation in rats. The acute IOP elevation protocol is employed to gauge the capacity of 58

the eye to cope with reduced OPP and increased mechanical stress. By comparing the 59

three BP groups, we aimed to differentiate the effect of chronic hypertension from acute 60

BP elevation per se. More specifically, we hypothesized that despite an improvement in 61

OPP, the ERG and ocular blood flow will be more susceptible to IOP elevation in chronic 62

hypertension. 63

Materials and methods 64

Animals 65

All animal experimental procedures were conducted in accordance with the ARVO 66

Statement for the Use of Animals in Ophthalmic and Vision Research. Animal ethics 67

approval was obtained from the Animal Ethics Committee (Ethics ID: 0705168) of the 68

University of Melbourne. Adult Long-Evans rats (8 - 12 weeks, 200 - 400 g) were housed 69

in a 20°C environment with light cycling (12-hour light/12-hour dark, on at 8 am, 50 lux 70

maximum). Food (Barastoc Rat and mice feed, Ridley Agriproducts, VIC, Australia) and 71

water were available ad libitum. 72

Experimental design 73

Chronic high BP was induced in 15 rats by subcutaneous infusion of Angiotensin II (Ang 74

II, 0.7 mg/kg/day, 2.5 µL/hour delivered by an Alzet® osmotic minipump, model 2ML4, 75

5

Durect Corporation, CA, USA) for 4 weeks. In the control group, 5 rats received vehicle 76

(normal saline) infusion for an equivalent volume and duration. Systolic BP and IOP were 77

monitored in conscious animals at baseline and then weekly throughout the four weeks. 78

At the end of four weeks, the effect of chronic hypertension induced by Ang II on retinal 79

function was assessed using the scotopic ERG recorded in response to a range of 80

stimulus intensities (see below for details). The effect of chronic hypertension on ocular 81

susceptibility to IOP challenge was considered by raising IOP in 5mmHg steps over ~1 82

hour (10 to 120 mmHg, 3 mins per step), during which ERG (retinal function) was 83

recorded in one eye and LDF (blood flow) in the fellow eye. ERG and LDF response to 84

IOP elevation was compared across the 3 groups: animals with four-week hypertension, 85

1-hour hypertension of the same magnitude, and normal controls. Data for the acute 86

hypertension group was reported previously3 and re-plotted here for comparison (with 87

permission from PLoS One). 88

Following the above experiments, animals were euthanized prior to dissection of the left 89

ventricle and aorta for gross morphology and histology to consider arterial and cardiac 90

morphological changes associated with chronic hypertension. 91

Blood pressure elevation and monitoring 92

In the chronic hypertension group, Ang II was delivered subcutaneously for 4 weeks by 93

implanting an osmotic minipump. For implantation, rats were anaesthetized with a 94

ketamine;xylazine mixture (intramuscular, 60:5 mg/kg). Then a 1 cm skin incision was 95

made on the rat’s back and an osmotic minipump was inserted into the subcutaneous 96

space. The wound was closed using interrupted sutures and caprofen was injected 97

(subcutaneous 4mg/kg) for post-surgical analgesia. 98

Systolic BP was monitored in conscious rats using a tail cuff sphygmomanometer 99

(ML125, ADIntruments Pty Ltd. NSW, Australia). Basal systolic BP was measured daily 100

for 5 days in conscious rats (n = 20) to return the 95% CI for mean habitual systolic BP. 101

6

During experiments, BP was measured three days in each week. Daily measurement 102

was, in turn, the average of 10 repeats over 10 minutes (1 min intervals). To control for 103

diurnal fluctuation, all BP measurements were taken between 10 am and 12 noon. After 104

gentle immobilization in a custom-made restrainer, animals were allowed to acclimatize 105

for 20 minutes to minimize stress-related changes in BP (supplementary materials, 106

Figure S1). Following the 20-min acclimatization, systolic BP was recorded every minute 107

for 10 minutes. 108

The effect of chronic hypertension on ERG and blood flow was compared with transient 109

high BP of similar magnitude, induced by intravenous infusion of Ang II for 1 hour (data 110

from previous study).3 In brief, 1% Ang II was infused via a femoral vein cannula at a rate 111

of 45 – 90 µg/kg/min. The infusion rate was adjusted as needed to maintain BP at a 112

stable level over the duration of the acute IOP elevation. 113

During acute IOP challenge, BP was measured directly via femoral artery cannulation in 114

all groups. MAP was calculated as [MAP = diastolic + 1/3 (systolic – diastolic BP)]. 115

Acute IOP challenge 116

IOP was elevated from 10 to 120 mmHg in steps of 5 mmHg, each lasting 3 minutes 117

(total duration of 69 minutes). During LDF measurement, IOP elevation was induced by 118

anterior chamber cannulation using a 30 gauge needle connected to a height adjustable 119

reservoir containing normal saline. The height of the fluid level was calibrated against a 120

mercury manometer to produce the desired IOP. During ERG measurements, 121

cannulation was performed in the vitreous chamber to avoid interference with ERG 122

electrodes placed on the cornea. We have previously shown that both anterior and 123

posterior chamber cannulations produce the same IOP elevation as verified by an 124

independent measurement cannula (see online supplement of He et al, 2012).3 125

7

Electroretinography 126

Following minipump implantation, the scotopic ERG was measured weekly under 127

general anesthesia (intramuscular ketamine 60 mg/kg, xylazine 5 mg/kg). Following 128

dark-adaptation for 12 hours, one drop of 0.5% proxymetacaine and one of 0.5% 129

tropicamide was applied for topical anesthesia and for mydriasis, respectively. A 130

Ganzfeld sphere (Photometric Solutions International, VIC, Australia) was used to 131

delivery luminous energy ranging from -6.26 to 2.07 log cd.m-2.s calibrated with an 132

IL1700 integrating photometer (International Light Technologies, Peabody, MA, USA). 133

Responses were recorded using an active electrode (custom-made, chlorided sliver 134

electrode) placed on the corneal apex and referenced to a ring electrode placed behind 135

the limbus on the conjunctiva. A ground needle electrode was placed in the tail. Signals 136

were amplified (x1000) over a band-pass of 0.3 – 1000 Hz (-3dB) and digitized using an 137

acquisition rate of 4 kHz. 138

ERG waveforms were analyzed using well-established methods described in past 139

publications.12-14 In brief, the photoreceptor response was modeled to return amplitude 140

(RmP3, µV) and sensitivity (S, cd-1.m2.s-3), in terms of a delayed-Gaussian function15, 16 fit 141

to the leading edge of the a-wave. The rod bipolar cell function was also quantified in 142

terms of its maximal amplitude (Vmax, µV) and sensitivity (1/K, cd-1.m2.s-1), from the 143

modeling the b-wave amplitudes as a function of stimulus energy with a hyperbolic 144

curve.17 Ganglion cell response was measured as the peak-to-trough amplitude of the 145

Scotopic Threshold Response (STR) recorded at -5.25 log cd.m-2.s. 146

During the step-wise IOP elevation, a single ERG b-wave (stimulus energy -1.12 log 147

cd.m-2.s) was serially measured at the end of each IOP step. The signal was quantified 148

as the trough-to-peak amplitude. 149

Laser-Doppler flowmetry 150

During IOP elevation, ocular blood flow was monitored continuously at a sampling rate of 151

8

1 kHz using LDF (ML191, ADInstruments Pty Ltd, NSW, Australia). As previously 152

described,3, 18 a needle-type probe (0.48 mm diameter, MNP110XP, ADInstruments) was 153

inserted 2 mm into the vitreous chamber to measure blood flow changes in response to 154

IOP elevation. The backscatter returned by the LDF probe provided an indication of the 155

distance between the probe and the retinal surface. Such backscatter was kept at a fixed 156

level by a micromanipulator throughout blood flow measurement, thereby minimizing any 157

movement-related artifact due to IOP elevation. 158

Both retinal and choroidal blood circulations contribute to the signal returned by the 159

intravitreous LDF probe used in this study (please see online supplement of He et al, 160

2012).3 Although the exact proportion of the two components is not precisely known, a 161

further pilot study showed that at least 50% of the LDF signal is derived from the retinal 162

circulation (supplementary materials, Figure 2S). Given that photoreceptor oxygenation 163

is derived from the choriocapillaris and inner retinal function is dictated by the retinal 164

circulation, this combined measure is an appropriate index of ocular blood supply. 165

Therefore throughout this manuscript the term “ocular blood flow” is employed to 166

describe the collective contribution from both circulations. 167

Morphology of heart and aorta 168

At the end of experiments, animals were euthanized prior to dissection of the heart and 169

aorta. Immediately post mortem, the heart was excised, cleared of blood and the 170

thickness of the left ventricle was measured at 1 mm from the cardiac apex with a vernier 171

caliper (Mitutoyo Corporation, Japan). This procedure eliminates any shrinkage 172

produced by paraffin embedding. Each measurement was an average of 4 repeats. 173

The left ventricle-septum complex was dissected under a 10X microscope at anatomical 174

landmarks to improve consistency. After the tissue was freeze-dried for 2 hours 175

(Micromodulyo 1.5K Vacuum Freeze Dryer, Edwards High Vacuum International, UK), 176

the combined dry weight of the left ventricle and septum was measured and expressed 177

9

as a percentage of the body weight. 178

A 0.5 mm segment of the thoracic aorta at the level of diaphragm was dissected and 179

fixed in neutral buffered formalin (4% paraformaldehyde in phosphate buffer pH7.2) 180

overnight. Prior to sectioning, the tissue was dehydrated through graded alcohol, 181

transferred to xylene and infiltrated with molten paraffin wax. Using the standard fast 182

procedure,19, 20 the tissue was passed through three changes of 100% ethanol, xylene, 183

and paraffin. After embedding in paraffin blocks, aortic cross-sections of 10 µm thickness 184

were cut and mounted on glass slides. Sections were dewaxed, rehydrated and 185

prepared for for Gomori’s Aldehyde-Fuchsin staining. To enhance visibility of elastin 186

fibres, Indigo Carmine-picric Acid was applied as a counterstain. Light microscopy and 187

image capture was performed at 10 times magnification using a DotSlide 2.0 Whole 188

Slide Scanner (Olympus BX4, Japan). To avoid bias, image analysis (software 189

Image-Pro Plus 6.0, Meda Cybernetics, USA) was conducted by two independent 190

investigators who were masked to the identity of blood pressure status. As described 191

previously,21, 22 the wall-to-lumen ratio was defined as the area of the media tunica 192

relative to that of the lumen. The sum of Aldehyde-Fuchsin stained area was quantified 193

as elastin content and expressed as a percentage of total tissue area (ie. elastin + 194

collagen). 195

Sample size and data analysis 196

When comparing chronic and acute high blood pressure, it is important that both groups 197

have similar BPs (~190 mmHg) during the IOP challenge protocol. Therefore, only 9 of 198

the 15 eyes in the chronic high blood pressure group were used in the ERG assay. Of 199

the 6 eyes that were excluded, 3 were due to excessively low BP arising from general 200

anaesthesia, and 3 due to surgical complications (e.g. fluid leakage, cataract formation). 201

Likewise, when LDF was measured in the fellow eyes, 11 of the 15 eyes had BP levels 202

under anaesthesia that were comparable to those achieved in the acute hypertension 203

group. 204

10

Results 205

Blood pressure and IOP 206

Five daily habitual systolic BP and the 95% CI for mean (108 – 119 mmHg) are shown in 207

Figure 1A. In Figure 1B, systolic BP at week 0 represents average baseline 208

measurements for each group prior to treatment. With Ang II infusion, systolic BP 209

increased steadily to levels well above baseline (192 ± 4 mmHg by week 4, p < 0.05). In 210

controls that received normal saline infusion, systolic BP remained within the 95% CI of 211

baseline (p > 0.05). Despite the marked BP difference, IOP was similar between the two 212

groups. (Figure 1C, two-way RM ANOVA, interaction term p = 0.97, between groups p = 213

0.78). There was significant IOP fluctuation over the four weeks in both groups (time 214

effect p < 0.001). 215

216

Figure 1. BP and IOP profile measured in awake rats with chronic hypertension induced by subcutaneous

infusion of Ang II over 4 weeks. A: baseline systolic BP measured daily over five consecutive days (n =

20) which returns the 95% confidence interval for baseline systolic BP (shaded area). B: The cohort in A is

divided into chronic hypertension (n = 15, filled symbols) and sham control (n = 5, unfilled symbols). BP

at week 0 represents the average of the five baseline measurements in Panel A. C: IOP in hypertensive rats

and sham controls, symbols jittered along x-axis for clarity. Error bars represent standard error of mean.

217

11

Cardiovascular consequences of chronic hypertension 218

After 4 weeks, the body weight of the hypertensive rats was similar to controls (Figure 2A, 219

310 ± 8 vs 321 ± 13 g, p = 0.481, t-test). Hypertensive rats developed cardiac 220

hypertrophy, as evidenced by the increased left ventricle-septum complex dry weight 221

(0.15 ± 0.01%) relative to body weight compared with controls (0.10 ± 0.01%, p = 0.017, 222

t-test). There was also a trend for the left ventricle to be thickened in chronic 223

hypertension, though this difference just failed to reach statistical significance (1.91 ± 224

0.11 vs 1.51 ± 0.06 mm, p = 0.058, Figure 2C). 225

In addition to cardiac hypertrophy, hypertensive rats also developed structural change in 226

the aorta. In Figures 2D and 2E, the cross-section of the aorta (Gomori’s Aldehyde 227

stained, purple: elastin; light blue: collagen) shows a thicker layer of tunica media 228

(smooth muscle) in the hypertensive rat. Group analysis confirmed that both aortic wall 229

thickness (p = 0.017, Figure 2F) and wall-to-lumen ratio (p = 0.018, Figure 2G) were 230

greater in chronic hypertensive rats. Elastin content relative to total tissue (elastin / 231

elastin + collagen, %) was not significantly different between the two groups (p = 0.812, 232

t-test, Figure 2H). 233

12

Figure 2. Body weight, cardiac hypertrophy and increased aortic wall thickness in rats with chronic high

blood pressure (filled bars). (A) Chronic hypertensive rats (n = 15) have the same body weight as controls

(n=5). (B) Chronic hypertension returns cardiac hypertrophy as evident by the increased dry weight of the

left ventricle and septum, (C) when compared with normotensive rats (n = 5) the slight increase in left

ventricle thickness seen in the hypertensive group just failed to reach significance (p=0.06). Histology of

aortic cross-section (D & E) showed increased wall thickness and wall-to-lumen ratio (quantified in F &

G), without change in the proportion of elastin content (H). Gomori-Aldehyde Fuchsin stain: purple for

elastin, light blue for collagen. Asterisk indicates statistical significance (p < 0.05). Error bars represent

standard error of mean.

234

Effect of 4-weeks of hypertension on retinal function 235

Figure 3 compares retinal function between chronically hypertensive and normotensive 236

rats. Representative ERG waveforms across a range of stimulus intensities suggest little 237

difference between the two groups (Figure 3A). Quantifying the components of 238

13

photoreceptor amplitude (RmP3) and sensitivity (S), bipolar cell amplitude (P2 Vmax) and 239

sensitivity (1/k), as well as ganglion cell amplitude (STR) confirms that after 4 weeks of 240

hypertension, responses in chronic hypertension were comparable to control animals 241

(Figure 3B, two-tail unpaired t-test, p = 0.962, 0.086, 0.919, 0.111, 0.510 respectively). 242

243

Figure 3. Effect of 4-week hypertension on retinal function measured using the electroretinogram (ERG).

A: Representative ERG waveforms recorded over a range of stimulus intensities in hypertensive (black

traces) and nomotensive (grey traces) rats 4 weeks after minipump implantation. B: Photoreceptor, bipolar

cell and ganglion cell parameters were similar between chronic hypertensive rats (filled bars) and controls

(unfilled bars) (two-way ANOVA, p > 0.05 for both interaction term and between groups). Error bars

represent standard error of mean.

244

Susceptibility to acute IOP elevation 245

Retinal susceptibility to a physiological stressor was considered by measuring the ERG 246

and LDF during IOP elevation and has been shown normalized to baseline in Figure 4. 247

For both relative retinal function (b-wave amplitude) and blood flow (LDF), the 248

IOP-response curves show a rightward shift in hypertensive rats when compared with 249

controls (two-way RM ANOVA, interaction p < 0.001 for Figure 4A and p = 0.002 for 250

14

Figure 4B), consistent with a reduced susceptibility to IOP elevation. 251

The chronicity of hypertension (chronic vs acute) was considered in animals matched for 252

blood pressure (for ERG assay, MAP 161 ± 13 vs 161 ± 4 mmHg, p = 0.997; for LDF 253

assay, 155 ± 13 vs 156 ± 5 mmHg, p = 0.944, t-test). Figures 4A and 4B show that the 254

degree of protection against IOP elevation was reduced in chronically hypertensive rats 255

compared with acute hypertensive animals. A two-way RM ANOVA comparing 256

IOP-response curves between the two forms of hypertension returns a significant 257

interaction term when IOPs greater than 60 mmHg are considered (Figure 4A p = 0.003, 258

Figure 4B p < 0.001). Thus 4-weeks of chronic hypertension compromise the amount of 259

protection afforded by the increase in OPP resulting from the higher blood pressure. 260

261

Figure 4. Effect of chronic hypertension on the susceptibility of retinal function and ocular blood flow to

acute IOP challenge. A: IOP-induced retinal dysfunction in acutely hypertensive, chronically hypertensive

and normotensive rats. B: IOP-induced changes in blood flow in acutely hypertensive, chronically

hypertensive, and normotensive rats. Data for acute hypertension is reproduced from our previous work

(with permission from PLoS One).3 Error bars represent standard error of mean.

262

15

Discussion 263

Consistent with our previous4 and other studies of acute hypertension,1-4 chronic 264

hypertension of 4 weeks duration makes neuronal function and ocular blood flow less 265

susceptible to acute IOP elevation as indicated by a rightward shift of the 266

IOP-response relationship when compared with normotensive rats (Figure 4A). By 267

matching the blood pressure levels in acute and chronically hypertensive rats we 268

showed, for the first time, that 4-weeks of chronic hypertension partially negates this 269

effect. 270

To date, few experiments have considered the effect of chronic hypertension on the 271

susceptibility to IOP elevation. The neural response was normal under basal conditions 272

(no IOP stress), but was altered when challenged with IOP elevation. In monkeys, 273

Hayreh and colleagues23-25 investigated the influence of arterial hypertension on optic 274

nerve head damage induced by chronic IOP elevation. The strength of their study was 275

that long-standing hypertension (40 – 90 months) and atherosclerosis (62 – 105 months) 276

was induced before IOP was elevated (4 – 60 months) by laser photocoagulation of the 277

trabecular meshwork. Such a long period of experimentation better models the chronicity 278

of human hypertension and the subsequent development of glaucoma. These authors 279

found that systemic hypertension / atherosclerosis produced either no effect23, 25 or only 280

a marginal exacerbation24 of optic nerve head and retinal nerve fibre layer changes in 281

their model of chronic IOP elevation. Given the substantial variability in duration and 282

magnitude of BP and IOP elevations in their cohort, the authors acknowledged that their 283

study might have been underpowered to detect a difference. Nevertheless, their finding 284

is consistent with our observation for the relative detrimental effect in animals with 285

4-week chronic hypertensive. 286

The reactivity of arterial wall is crucial to mechanisms that preserve blood flow during 287

mild ischemic events (blood flow autoregulation).26 We found that 4 weeks of arterial 288

16

hypertension induces structural changes with thickened and narrowed arterial wall. This 289

data indicates that systemic or local vascular dysfunction in this pharmacologic rodent 290

model (Ang II) is sufficient to compromise autoregulatory mechanisms. We believe that 291

this modification of OPP-related benefits is independent of local effect on elements of the 292

renin-angiotensin system known to exist in the retina.27 Our reasoning is based on fact 293

that neither systemic Ang I nor Ang II is able to cross the blood-brain barrier28, 29 or the 294

blood-retinal barrier.30 Additionally, the infusion of Ang II is an often used model for 295

systemic hypertension in rodents.31-33 This approach mimics excessive renin-angiotensin 296

system activity, which is known to play an important role in the pathogenesis of essential 297

hypertension. In acutely hypertensive rats, it is understood that Ang II raises BP largely 298

through vasoconstriction and the resultant increased peripheral arterial resistance. In the 299

chronic case, Ang II acts as a vasoconstrictor, but it also stimulates aldosterone 300

production, leading to sodium and water retention by the kidney, with both factors 301

promoting high blood pressure. The time course and the extent of BP elevation in this 302

study shows agreement with previous works utilizing similar Ang II dosing in rats.34, 35 303

Our data suggests that the reduced neural capacity to withstand IOP elevation in the 304

chronic hypertension (Figure 4A) is associated with a relatively impaired blood flow 305

autoregulation (Figure 4B). Whilst the exact mechanism by which chronic hypertension 306

impairs retinal autoregulation (reduced blood flow resistance to IOP) is not fully 307

understood, changes in the thickness of vessel walls, altered compliance and rigidity 308

have been implicated.36 In addition to its pressor effect to increase BP, Ang II is a potent 309

stimulator for cell growth and remodelling in cardiac myocytes and arteries.37 Similar to 310

the pathophysiology in essential hypertension, hypertensive rats in this study developed 311

ventricle hypertrophy and increased arterial wall thickness (Figure 2). However, vascular 312

remodelling was not evident, as the elastin/collagen ratio in the aorta, an indicator for 313

arterial compliance and rigidity,38 was not significantly changed at our 4-week time point 314

(Figure 2H). 315

17

Although cardiac and aortic hypertrophy were observed, it is not clear whether similar 316

changes also occurred at the level of the retina. One limitation of our study is that 317

structural changes in the retinal vasculature were not measured. Previous studies of the 318

same animal model showed that increased vascular thickness and rigidity are present 319

not only in the aorta, but also in the small arteries and arterioles of the rat mesentery.35, 39 320

Therefore, it may be reasonable to assume that the smooth muscle hypertrophy in the 321

aorta represents a more widespread change that involves the retinal and choroidal 322

vasculature. Such change may underlie the impairment in ocular blood flow 323

autoregulation we have observed here (Figure 4B). 324

Four weeks of chronic hypertension did not completely negate the beneficial effects 325

arising from greater OPP. This may be consistent with the early stage of hypertensive 326

vascular disease as evidenced by the absence of vascular remodeling (aortic 327

elastin/collagen ratio, Figure 2H). Also consistent with the idea of an early disease model 328

in this study, retinal function was unaltered in the chronically hypertensive rats (Figure 3). 329

Such finding differs from a number of previous studies where retinal dysfunction has 330

been reported in both patients with chronic hypertension40 and after prolonged 331

hypertension in rat models (Ren-2 rat,41 spontaneously hypertensive rats42). In those 332

studies ERG changes were associated with retinal vascular alterations. Thus any 333

structural changes in the retina vasculature in the current model are not severe enough 334

to compromise basal retinal function. We speculate that, with longer periods of 335

hypertension and thus more severe vascular damage, the beneficial effect of high BP 336

might be further reduced. 337

It is important to rule out that changes in sensitivity to IOP elevation do not arise from 338

altered habitual IOP levels. Previous studies have shown that BP elevation can cause a 339

modest increase in IOP. In humans, every 10 mmHg rise in Systolic BP is associated 340

with an IOP elevation of ~0.27 mmHg.10, 43-46 A similar association has been reported in 341

spontaneous hypertensive rats at 8 weeks of age.47 Based on this association, systolic 342

18

BP elevation from 110 to 190 mmHg reported in our study could raise IOP by ~2.2 343

mmHg, which is of little practical significance. As we found no significant difference in 344

IOP by 4-weeks, it is unlikely that differences in susceptibility to IOP elevation arise from 345

changes to habitual IOP. 346

A methodological issue in this study is that the precise location of blood flow 347

autoregulation is difficult to pinpoint as the LDF contains contributions from retinal and 348

choroidal circulations, which respond differently to IOP elevation (Zhi et al 2012). 349

Nevertheless, the difference in blood flow response between normotensive and chronic 350

hypertensive is robust, and correlate well with our retinal function measurement. Further 351

studies using techniques such as optical coherence tomography microangiography 352

would help differentiate autoregulatory deficiency in retina and choroid. 353

It is also important to note that any pharmacological approach to manipulate BP has the 354

potential to confound studies of vascular autoregulation. There are alternative, drug-free 355

approaches to elevate BP chronically, such as the spontaneous hypertensive rats, or the 356

kidney artery clipping model. However, with these methods, it would not be easy to 357

compare acute and chronic hypertension with common BP level. Whilst being aware of 358

the limitation of our Angiotensin II model in studying autoregulation, we chose the model 359

not only because it allows comparison between acute and chronic hypertension of the 360

same BP, but also it mimics excessive rennin-angiotensin activity, an important aspect in 361

the etiology of human hypertension. 362

363

364

Summary 365

In this study we showed that 4 weeks of chronic hypertension compromises the benefit 366

afforded by BP elevation for retinal function against IOP elevation. This effect was 367

19

partially associated with a reduced capacity for ocular blood flow to autoregulate in 368

response to IOP elevation in chronic hypertension. Structural changes to blood vessels 369

arising from chronic hypertension may underlie some of our observations but longer 370

interventions are needed to evoke the long term components (vascular and neural) of 371

this response. 372

Acknowledgements 373

Mr Jonathan Bensley and Ms Debbie Arena from the Department of Anatomy and 374

Developmental Biology, Monash University, assisted in the histology assay and analysis. 375

The study is funded by the NHMRC Project grant (566570, 1046203)376

20

Supplementary materials 377

Figure 1S Minimizing stress-related BP response associated with conscious recording. A: restraining tube

used for tail cuff sphygmomanometer in a conscious rat. B: in 20 rats, systolic BP (mean ± SEM) was

repeatedly measured at 1 minute intervals for 35 minutes. The BP change over this period was biphasic

and described by a two-line model: a decline during the initial 14.7 min (95% CI 10.5 – 18.8) followed by

a plateau where the slope does not significantly differ from 0 (-0.21, 95% CI -0.56 – 0.14). We adopted a

time window of 20 – 30 min (shaded region) to measure BP to minimize stress-induced BP artifact.

21

Figure 2S. Contribution of retinal blood flow to the laser-Doppler flowmetry (LDF) in rats. Blood flow is

measured with LDF probe inserted in the vitreous chamber while retinal circulation in a blood vessel under

the probe is transiently blocked. A & B: fundus views before and during vascular occlusion induced by

insertion of a blunt occluder (lower probe) while maintaining the position of (upper) LDF probe. Scale bar

= 200 µm The arrow heads indicate the area of blood vessel occlusion. C: blood flow in this eye is halved

during vascular occlusion for 10 seconds, whilst the backscatter (D) remains stable indicating a constant

measurement distance. E & F: the group data (n = 6) binned in 2-second intervals and expressed relative to

baseline. The result suggests that at least 50% of the LDF signal is attributable to inner retinal circulation.

Shaded boxes: duration of vascular occlusion. Error bars: SEM. Care should be taken in interpreting these

22

data as the LDF measurement includes the first order blood vessel which is approximately 40 micron in

diameter (Panel A). Blood vessel of this size may come close to violating the Bonner-Nossal theory48 upon

which the LDF is based.

378

23

379

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References 380

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