Enhanced Physiological Stress Response in Patients with Normal Tension Glaucoma during Hypoxia

Abstract

Purpose. To investigate whether patients with normal tension glaucoma (NTG) show an enhanced stress response to reduced oxygen supply compared to age-matched healthy controls, measured by serum adrenaline and endothelin-1 (ET-1) levels and changes in distal finger temperature. Methods. A thorough clinical characterization of patients with NTG and age-matched controls was performed prior to inclusion in the study. Twelve patients with NTG and eleven healthy controls met the inclusion criteria and were enrolled in the study. All subjects underwent a two-day investigation. Participants were randomly exposed to either hypoxia or normoxia during the first visit. Hypoxia or normoxia was induced for two hours through a tightly fitting face mask. In addition, the peripheral circulation was assessed with a thermographic camera. Blood samples were obtained before, during, and after hypoxia or normoxia to evaluate systemic stress molecules such as catecholamines and ET-1 levels. Results. In patients with NTG, reduced oxygen supply induced an increase in peripheral blood adrenaline ($p$ < 0.05) and a decrease during recovery ($p$ < 0.01). A difference in distal finger temperature was shown in patients with NTG under hypoxia compared to normoxia (exposure: $p$ < 0.05; recovery: $p$ < 0.05). Hypoxia induced an increase in peripheral blood ET-1 levels in both groups (NTG: $p$ < 0.01; controls: $p$ < 0.05). Conclusion. Patients with NTG had an enhanced physiological stress response as a consequence of hypoxia compared with age-matched controls. Although more studies are needed, the present study supports the involvement of vascular risk factors in the pathophysiology of NTG.

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1. Introduction

Glaucoma is a progressive optic neuropathy characterized by loss of retinal ganglion cells (RGC) and their axons. Along with the loss of RGC, the disease is accompanied by a gradual loss of the peripheral visual field [1–4]. Glaucoma is the most frequent cause of incurable blindness and is estimated to affect approximately 111.8 million people by 2040 [5]. Together with aging, increased intraocular pressure (IOP) is recognized as the most important risk factor. However, patients may have an IOP in the normal range and still develop glaucomatous progression (normal tension glaucoma, NTG) [6]. Therefore, although the only existing treatments for glaucoma are IOP-lowering strategies, glaucoma is a multifactorial disease with many different risk factors [7]. Thus, identifying and characterizing other instigators are essential.

An increasing number of studies have suggested that glaucoma is a systemic disease that manifests in the inner retina, resulting in loss of RGC. The reason for this particular RGC vulnerability is the fact that these cells are especially dependent on a constant oxygen sand energy supply [8–12].

Consistent with this hypothesis, an increasing number of studies have shown that there is an association between vascular dysfunction and NTG [8–10, 12–14]. Vascular dysfunction is therefore thought to be an important risk factor for onset and progression of NTG [8–12, 15, 16].

Vascular dysfunction is defined as a condition in which the actual blood flow does not meet the requirements of the tissue for oxygen supply [13, 17]. This can result in overperfusion or underperfusion, which is basically due to an imbalance in the relationship between molecular vasoconstrictors and vasodilators. Dysregulated vascular constriction or dilation of the retinal blood flow may inevitably cause periods of hypo- and hyperperfusion [18–20], further escalating to hypoxic events and oxidative stress. As a result, such a cascade of reactions may contribute to glaucomatous neurodegeneration [11, 12, 21–25]. The body stabilizes blood flow by a highly regulated release of vasoconstrictive and vasodilative molecules [8–11]. This tight regulation of the vessel dynamics is termed autoregulation [18] and maintains flow by altering vessel diameter in response to changes in perfusion pressure [8, 12, 13]. Multiple factors influence autoregulation, such as CO₂ levels, temperature, low grade inflammatory molecules, catecholamines, and ATP production [8, 9, 13, 15, 26]. Many of these variables are influenced by hypoxia and have been explored in animal experimental models of glaucoma [27–29]. However, to our knowledge, the relationship between decreased oxygen supply and these molecular changes has not been studied in patients with glaucoma.

Thus, the present study aimed to provide novel insight into systemic effect and molecular changes in response to reduced oxygen supply in patients with glaucomatous neurodegeneration compared to controls. Since we assume that all patients with glaucoma have multiple risk factors and since we were particularly interested in studying IOP-independent factors, we examined glaucoma patients with IOP within the normal range, where IOP is hypothetically a less significant risk factor. We thus examined patients with NTG and compared their response to systemic hypoxia with age-matched control subjects. We have previously shown that hypoxia reduces oxygen saturation levels in healthy test subjects and in patients with NTG within six minutes when they breathe 10 % oxygen [30]. With this human experimental model, hypoxia is used as a universal oxygen stress model. In the present study, we aimed to investigate whether patients with NTG have an enhanced stress response compared to healthy controls when exposed to reduced oxygen availability, including measuring various vascular parameters such as serum levels of catecholamines and ET-1 as well as distal finger temperature.

2. Methods

A total of 23 eligible test subjects participated in the study between May 2015 and August 2016 [30]. The test subjects were assigned into two different groups: patients with NTG (12 participants) and age-matched healthy controls (11 participants) (Table 1 and Figure 1). Power and sample size calculations were based on an arterial vessel diameter from a previous study by Wong et al. [31]. In this study, the mean arterial vessel diameter was found to be 204.4 μm with a standard deviation of 18.6. With a power of 80%, a $p$ value of 0.05, and an allowed variation in diameter of 12%, a total of 9 individuals were required in each group. Other published works that required gas inhalation of volunteers have used similar sample size to that in this study [32]. All participants were ≥50 years old and nonsmokers. Patients with NTG were recruited by a glaucoma specialist through the Department of Ophthalmology at Zealand University Hospital, Roskilde. Control subjects were recruited via opticians to the study “Detecting Visual Fields Defects With Damato Multifixation Campimetry Online” [33] and were invited to participate in the present study.

Table 1

Characteristics of participants.

	NTG	Controls	$p$ value
Age	70.3 ± SEM 1.5	65.6 ± SEM 2.3	0.09
Gender	Female: 6 (50%)Male: 6 (50%)	Female: 5 (45%)Male: 6 (55%)	0.83
BMI	23.6 ± SEM 1.0	25.8 ± SEM 0.8	0.11

[figure omitted; refer to PDF]

This interventional case-control study was performed in compliance with the Declaration of Helsinki approved by the National Committee on Health Research Ethics (ethical protocol: H-2-2014-060). All participants received written information about the study and had the study verbally explained and provided both oral and written consent prior to participation. Inclusion criteria and exclusion criteria are summarized in Tables 2 and 3.

Table 2

Inclusion criteria for patients with NTG.

Untreated intraocular pressure (IOP) never detected higher than 21 mmHg measured at different times of the day (8 AM–5 PM)

Open anterior chamber angles observed by gonioscopy

Glaucomatous cupping characterized by a violated ISNT rule (that normal eyes show a characteristic configuration for disc rim thickness of inferior ≥ superior ≥ nasal ≥ temporal)

Glaucomatous visual field loss by Humphrey perimetry or Octopus perimetry

Table 3

Exclusion criteria for patients with NTG and healthy age-matched controls.

Medical history including ocular trauma or eye conditions other than glaucoma involving the optic nerve

Significant systemic disease, e.g., hypertension, heart failure, hypercholesterolemia, diabetes mellitus, autoimmune diseases, and previous cerebral infract or bleeding

Individuals who were unable to cooperate during examination

Individuals below the age of 50 years

Individuals who smoke

All subjects underwent two days of investigation. In random order, the visits included either normobaric hypoxia or normobaric normoxia. Hypoxia/normoxia was induced for two hours through a tight fitting face mask. The mask was connected via a Y-piece to a Douglas bag which was filled with either atmospheric air or a mixture of 10% oxygen and 90% nitrogen. We chose two hours of hypoxia induction to ensure a sustained effect on the cardiovascular system. This has been shown to be evident two hours after initial exposure [34]. Furthermore, two hours of hypoxia has previously been used to investigate endothelial function in healthy adults [35]. Previous studies have verified that the acute effect of hypoxia is abolished after 15 min, and we added a safety margin of further 15 min [36], resulting in a defined recovery period of 30 min after terminated hypoxia.

To ensure the safety of our participants, they were continuously monitored on both days of investigations with a three-lead ECG (M1166A model 66S, Hewlett Packard, Palo Alto, California, USA) and noninvasive blood-pressure measurement and pulse oximeter by a Nexfin monitor (BMEYE B.V., Amsterdam, Netherlands). We also encouraged our participants to let us know if they felt any discomfort. As an additional safety measure, the investigations were carried out at the Department of Anesthesiology, Rigshospitalet, Denmark, where we had anesthesiologists on call.

The two days of investigation were at least three weeks apart. All investigations were preceded by 12 hours of fasting.

2.1. Blood Samples

Blood samples were collected from a peripheral vein on both days of investigation before, during, and after hypoxia/normoxia as shown in Figure 2. At each collection, 3 × 6 mL EDTA glasses were taken and put on ice. The glasses were centrifuged for 10 minutes at 4000 rpm, after which the supernatant was pipetted to Eppendorf tubes and frozen at −80°C until further analyses.

[figure omitted; refer to PDF]

Despite the lack of autonomic innervation in the intraocular vascular system [44], it is likely that the systemic effects of adrenaline and noradrenaline play a role in the pathogenesis of glaucoma indirectly, if not directly [45–47]. In this context, Fitzgerald reports that systemic stress may lead to increased IOP [45]. In line with such systemic affection on retinal neurodegeneration, Horwitz et al. found that antiadrenergic antihypertensive drugs have a protective effect on the development of glaucoma [46, 47], which hypothetically may be explained by increased ocular perfusion. Thus, systemic levels of catecholamines may indirectly affect oxygen and energy supply to the inner retina and thereby play a role in the pathogenesis of glaucomatous neurodegeneration.

Since retinal vessels are autoregulated by ET-1 amongst others [8, 9, 13, 15, 26], we measured ET-1 in peripheral blood as a surrogate for the retinal ET-1 concentrations. ET-1 is a well-known vasoconstrictor in the eye and has repeatedly been demonstrated to play a key role in the regulation of ocular perfusion and hypothetically in the overall pathogenesis of inner retinal diseases [15, 48–51]. Circulating ET-1 can reach vessels in the optic nerve head (ONH) in two ways: (1) diffusion from the fenestrated choriocapillaris bypassing the blood-brain-barrier (BBB)/blood retinal barrier (BRB) or (2) through disrupted BBB/BRB. Disrupted BBB/BRB occurs both physiologically with aging and in response to neurodegeneration [15, 52, 53]. As a consequence of disrupted BBB/BRB, ET-1 can potentially freely access the vasculature supplying the optic nerve, and an increase in ET-1 in peripheral blood will lead to an increase in ocular ET-1 [15]. Our study showed a significant hypoxia-induced increase in serum ET-1 levels in both patients with NTG and controls (Figure 6). Previous studies have identified differences in ET-1 levels, when comparing patients with NTG to healthy controls. Li et al. have analyzed seven studies in a meta study, which showed higher plasma levels of ET-1 in the NTG group (mean difference of 0.6  pg/mL [$p$ = 0.007, 95% CI = 0.17–1.04]) [14]. However, the present study was not able to replicate these findings, possibly due to deviations in recruitment criteria concerning clinical phenotyping, ethnicity of the test subjects, and so forth.

In summary, the present study introduces the concept of an enhanced hypoxia-induced stress response in patients with NTG which may be correlated to glaucomatous neurodegeneration. However, future studies are required to evaluate retinal vessel diameter in response to hypoxia and correlate findings to other blood stress markers to elaborate on vascular dysfunction and hypoxia-mediated stress responses in patients with glaucoma.

Authors’ Contributions

Line Marie Dalgaard and Jeppe Vibæk contributed equally to this work.

Acknowledgments

The study was supported by Lions Prize, Fight for Sight Denmark, the Synoptik Foundation, the Michaelsen Foundation, the Danish Glaucoma Patient Organization, and the Novo Nordisk Foundation. Rupali Vohra is a part of the BRIDGE–Translational Excellence Programme (bridge.ku.dk) at the Faculty of Health and Medical Sciences, University of Copenhagen, funded by the Novo Nordisk Foundation (Grant no. NFF18SA0034956).

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