Corresponding Author: Yuki Hashimoto, PhD. Department of Orthoptics, Faculty of Medicine, Fukuoka International University of Health and Welfare, Momochihama 3-6-40, Fukuoka 814-0001, Japan. Although autoregulation varies in every vascular system, retinal vessels are more autoregulatory than choroidal vessels [1]. Changes in ocular perfusion pressure (OPP) have been shown to increase retinochoroidal blood flow immediately after dynamic exercise; however, this increase in blood flow was more sustained in the choroid-retina than in the retina. This difference may be related to the stronger blood flow autoregulation mechanism in the retina compared to that in the choroid [2]. Furthermore, it has been reported that blood flow in the optic nerve head (ONH) is autoregulated in response to fluctuations in intraocular pressure (IOP) and systemic blood pressure (BP) [3].
Cold pressor tests assess sympathetic responses in systemic circulatory dynamics [4-8]. This examination is used for screening hypertension and can predict future hypertension based on cardiovascular response [4,5]. Recently, a study that examined the vascular reactivity of the retina and choroid using functional optical coherence tomography (OCT) found that the vascular perfusion density of healthy individuals after a cold pressor test decreased in the choroid but remained unchanged in the retina [6]. Additionally, the choroidal morphology of healthy participants was quantitatively evaluated using enhanced depth imaging OCT and the cold pressor test; the results showed a significant decrease in subfoveal choroidal thickness (SFCT) after the test, and the decreased SFCT takes longer than other measures of circulation such as systolic BP (SBP), diastolic BP (DBP), mean BP (MBP), and OPP [8].
Laser speckle flowgraphy (LSFG) noninvasively measures the ocular blood flow and can quantitatively evaluate circulatory hemodynamics using the mean blur rate (MBR), which is a relative value of the blood flow velocity [9]. The wavelength of the diode laser used in LSFG is 830 nm (near-infrared light). The measurement light is irradiated onto moving erythrocytes within the retina and choroid. The resulting speckle pattern, formed by the interference of the scattered light, is then analyzed. To clarify, the faster the movement of erythrocytes, the lower the speckle contrast becomes [10,11]. The MBR is calculated from the change in the degree of blurring, which is proportional to the inverse square of the speckle contrast, and is used as a quantitative measure of relative blood flow velocity that has no units of quantity. If erythrocytes move faster, the speckle motion accelerates, resulting in increased blurring and an elevated MBR value. LSFG can create a two-dimensional color map based on blood flow velocity data, which is useful for confirming changes in the normal eye, monitoring the onset and progression of disease, and for follow-up observations [12-25]. The macular MBR reflects choroidal blood flow caused by the absence of the inner retinal layer at the fovea [12-21], whereas the ONH provides three MBRs: vessel average (MBR-V), tissue average (MBR-T), and overall average (MBR-A) [21-25].
Several studies have explored the hemodynamics of ONH using LSFG. The ONH blood flow has been reported to be highly autoregulated with its own diurnal variation [20], correlates with postmenstrual age in normal neonates [22], and does not differ between the left and right eye [23]. In Vogt-Koyanagi-Harada disease associated with anterior ischemic optic neuropathy, retinal blood flow velocity in the ONH decreases and choroidal blood flow velocity in the macula increases during the course of the disease, indicating that LSFG is a useful tool for evaluating the circulatory dynamics in the ONH and choroid [21]. Additionally, in patients with optic disc drusen, the blood flow velocity in the ONH is significantly reduced compared to that in normal eyes, and a significant correlation is observed between blood flow velocity and visual field defects [24]. Furthermore, in nasal optic disc hypoplasia patients (NOH), the MBR and circumpapillary retinal nerve fiber layer thickness ratios in NOH eyes significantly reduced compared to that in control eyes. Furthermore, a significantly positive correlation has been detected between MBR and circumpapillary retinal nerve fiber layer thickness in the nasal region [25]. Thus, LSFG is suitable for evaluating changes in the ocular hemodynamics of healthy individuals and those with various retinochoroidal diseases.
The autonomic nervous system (sympathetic nervous system) contracts blood vessels and increases BP in response to cold stimuli during winter [26,27]. The winter climate has been shown to affect the parameters associated with the increased risks of arteriosclerosis, vein occlusion, and hypertension [28,29]. In Japan, retinal vein occlusion (RVO) is a prevalent ocular disease with increasing incidence rates during the winter season, and hypertension is identified as one of its risk factors [30]. Furthermore, a study using LSFG has shown that in patients with RVO, the blood flow velocity of the ONH is reduced in the acute phase of the disease [31]. In addition, it has been reported that blood flow velocity in the ONH is also decreased in glaucoma. The meta-analysis revealed that MBR-V, MBR-T, and MBR-A are significantly decreased in glaucomatous eyes and there are significant differences in MBR based on the type of glaucoma [32]. Therefore, the consequences of these changes in ONH circulation dynamics may contribute to vascular autoregulation as a triggering factor for ocular disease.
We recently reported that macular MBR, an indicator of choroidal blood flow velocity, increases immediately after the cold pressor test [12]; however, changes in retinal vessel blood flow in the ONH as observed through LSFG after sympathetic nerve hyperactivity induced by the cold pressor test have not been assessed. During this study, we investigated vascular autoregulation of the ONH by measuring the time course of the change in the hemodynamics of the ONH after a cold pressor test using LSFG.
Materials and Methods
Ethics statement
This study was approved by the Ethics Committee of the Fukuoka International University of Health and Welfare (No. 20-fiuhw-022) and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all participants after the nature and possible consequences of the study were explained to them.
Study design and participants
This prospective study included 44 right eyes of 44 healthy volunteers (18 men and 26 women) with no ophthalmic, cardiovascular, or systemic diseases. Their mean age was 21.7 ± 5.0 years (range, 18-43 years). All participants had best-corrected visual acuity of ≥20 / 20. Each participant underwent examinations of the IOP, SBP, DBP, heart rate (HR), and LSFG. All measurements were conducted between 12:00 and 15:00 to account for the effects of diurnal variation [14,20], and participants refrained from smoking and engaging in exercise for at least 2 hours prior to the test and took rest for 10 minutes in a quiet room. In addition, these examinations were conducted at room temperature (24 ± 1 °C) with a humidity level of 47% ± 3% [12-15].
Cold pressor test
During the cold pressor test, the right hand was immersed up to the wrist in ice water with a temperature of approximately 1 °C for 30 seconds [6,8,12]. The IOP, BP, HR, and LSFG measurements were assessed at baseline, immediately after the test (0 minutes), and 10, 20, and 30 minutes after the test. At all measurement points, BP and HR were measured first, LSFG was performed second, followed by IOP testing. All examinations were performed with the participant in the sitting position, and each experimental session was completed within 3 minutes. Additionally, the participants were asked not to smoke or engage in exercise for at least 2 hours before the examinations, and they were allowed to rest for 10 minutes in a quiet room.
Retinal circulation measurements
The LSFG measurements were performed using the LSFG-NAVI (Softcare Ltd) to quantitatively assess the ONH blood flow velocity, and it has no unit of quantity. The LSFG results of all eyes were examined three times at each time point. A circular band was set on the color map of the ONH (Fig. 1A). At follow-up, each circle was automatically set using LSFG Analyzer ver. 3.7.0.4 (Softcare Ltd) at the same site where the circle was set at baseline. This software can evaluate MBR-V (white areas) and MBR-T (black areas), and MBR-A (overall average) separately using an automated definitive threshold. In order to evaluate the change in mean MBR-V, MBR-T, and MBR-A (Fig. 1B), we utilized the rates of change in each mean MBR value and compared them to the initial baseline values (defined as 100%) [12,13,16-19,22].
Hemodynamics
The IOP, BP and HR of all participants were measured at baseline, immediately after the cold pressor test, and 10, 20, and 30 minutes after the test. The IOP measurement was performed with a noncontact tonometer (NT-530, Nidek Co Ltd). Hemodynamics of the eye depend on the BP and IOP and are measured based on an indicator called OPP, which reflects systemic circulatory dynamics. The OPP was calculated using the IOP and BP values. The MBP was calculated using the SBP and DBP.
Statistical analysis
The Friedman test and Scheffé test were performed to examine sequential changes in the IOP, SBP, DBP, MBP, HR, OPP, and MBRs (MBR-V, MBR-T, and MBR-A). The same statistical analyses were also performed separately for men and women groups. For all tests, p < 0.05 indicated statistical significance.
Results
IOP, HR, and OPP data
Changes in the IOP and other systemic factors are summarized in Table 1 and Supplementary Tables 1 and 2. The IOP and HR did not change throughout the course of the study (Friedman test; IOP, p = 0.273; HR, p = 0.416). The changes in the OPP are presented in Table 1 and Supplementary Table 1. The mean OPP was 40.3 ± 5.0 mmHg at baseline, 44.3 ± 5.5 mmHg at 0 minutes, 40.9 ± 4.8 mmHg at 10 minutes, 40.8 ± 4.9 mmHg at 20 minutes, and 41.1 ± 5.3 mmHg at 30 minutes after the cold pressor test. The mean OPP value at 0 minutes was significantly higher than the baseline value (p < 0.001); however, no significant difference was observed at 10, 20, and 30 minutes relative to the baseline data. Separate investigations of the male and female groups (Friedman test) showed no significant changes in IOP (male group, p = 0.111; female group, p = 0.399) and HR (male group, p = 0.605; female group, p = 0.575), while OPP was significantly elevated only immediately after cold stimulation in both the groups (male group, p = 0.002; female group, p = 0.005) (Tables 2, 3).
SBP, DBP, and MBP data
The changes in the SBP, DBP, and MBP are presented in Table 1 and Supplementary Table 1. The mean SBP was 108.0 ± 10.6 mmHg at baseline, 113.9 ± 11.1 mmHg at 0 minutes, 108.0 ± 10.7 mmHg at 10 minutes, 107.5 ± 11.0 mmHg at 20 minutes, and 108.1 ± 10.5 mmHg at 30 minutes after the cold pressor test. The mean SBP value at 0 minutes was significantly higher than the baseline value (p < 0.001); however, there was no significant difference at 10 minutes (p = 0.998), 20 minutes (p = 1.000), and 30 minutes (p = 1.000) relative to the baseline data. The mean DBP was 66.2 ± 7.1 mmHg at baseline, 72.0 ± 8.3 mmHg at 0 minutes, 66.7 ± 6.7 mmHg at 10 minutes, 66.7 ± 7.0 mmHg at 20 minutes, and 67.1 ± 7.7 mmHg at 30 minutes after the cold pressor test. The mean DBP value at 0 minutes was significantly higher than the baseline value (p < 0.001); however, no significant differences were observed at 10 minutes (p = 1.000), 20 minutes (p = 0.997), and 30 minutes (p = 0.993) relative to the baseline data. The mean MBP was 80.2 ± 7.8 mmHg at baseline, 85.9 ± 8.3 mmHg at 0 minutes, 80.5 ± 7.4 mmHg at 10 minutes, 80.3 ± 7.5 mmHg at 20 minutes, and 80.8 ± 8.2 mmHg at 30 minutes after the cold pressor test. The mean MBP value at 0 minutes was significantly higher than the baseline value (p < 0.001); however, no significant differences were observed at 10 minutes (p = 0.999), 20 minutes (p = 0.962), and 30 minutes (p = 0.987) relative to the baseline data. In addition, the male group demonstrated significant increases in DBP (p = 0.021) and MBP (p = 0.016), whereas the female group exhibited significant elevations in SBP (p < 0.001), DBP (p = 0.039), and MBP (p = 0.005) only immediately after the cold stimulus (Tables 2, 3).
Changes in the MBR-V, MBR-T, and MBR-A of the ONH
The changes in MBRs are shown in Table 1 and Supplementary Table 1. The average MBR-V values of the ONH at baseline, immediately after the cold pressor test (0 minutes), and at 10, 20, and 30 minutes after the test were 44.3 ± 5.1, 44.3 ± 4.9, 45.2 ± 5.2, 44.6 ± 5.3, and 45.1 ± 5.4, respectively. The average MBR-T values of the ONH at baseline, immediately after the cold pressor test (0 minutes), and at 10, 20, and 30 minutes after the test were 13.3 ± 2.3, 13.4 ± 2.2, 13.6 ± 2.2, 13.3 ± 2.1, and 13.5 ± 2.0, respectively. The average MBR-A values of the ONH at baseline, immediately after the cold pressor test (0 minutes), and at 10, 20, and 30 minutes after the test were 25.0 ± 3.4, 24.8 ± 3.4, 25.1 ± 3.4, 25.1 ± 3.4, and 25.3 ± 3.2, respectively. The MBR values of the ONH did not change throughout the course of the study (Friedman test: MBR-V, p = 0.447; MBR-T, p = 0.258; MBR-A, p = 0.750). Additionally, MBR-V, MBR-T, and MBR-A did not change significantly during the course of the study for either the male group (Friedman test; p = 0.138, p = 0.157, and p = 0.930, respectively) or the female group (Friedman test; p = 0.852, p = 0.873, and p = 0.707, respectively) (Tables 2, 3).
Discussion
In the present study, we used as a quantitative measured changes in the MBRs of the ONH after the cold pressor test using LSFG. The SBP, DBP, MBP, and OPP increased significantly immediately after the test; however, no further changes were observed after 10 minutes. In contrast, the retinal blood flow velocity in the ONH did not change throughout the course of the study. The results were almost similar for the investigation of male and female groups when analyzed separately.
The diurnal variations of the macular MBR of choroidal blood velocity are significant, with the peak at 6:00 pm, when the sympathetic nervous system is active [14,20], and the MBR is significantly positively correlated with DBP, MBP, and OPP [20]. Recently, it was observed that healthy women with normal menstrual cycles had a more active sympathetic nervous system during the luteal phase compared with that during the follicular phase, suggesting that macular MBR increases in response to increased systemic hemodynamics, and the MBR exhibit a significant positive correlation with changes in DBP and MBP [19]. In addition, the sympathetic activity caused by physiological and psychological stress has been suggested to increase in winter, and choroidal blood flow velocity increases with increased IOP, BP, and OPP [15]. Furthermore, the macular MBR significantly increased by approximately 10% immediately after the cold pressor test, and a significant positive correlation is observed between the MBR and the SBP, MBP, and OPP [12].
However, interestingly, dynamic exercise changes the OPP and produces increased tissue blood flow to the retina during the immediate postexercise period, whereas blood flow increases more persistently in the choroid-retina [2]. It has also been reported that the ONH blood flow has its own diurnal variation because of strong autoregulation, whereas the choroidal blood flow is more likely affected by systemic circulatory factors because of poor autoregulation [20]. A recent study that examined the vascular reactivity of the retina and choroid using functional OCT showed that the vascular perfusion density of healthy individuals after the cold pressor test decreased in the choroid but remained unchanged in the retina. Furthermore, the choroidal vascular perfusion density has a strong inverse correlation with integrated muscle sympathetic nerve activity [7]. Additionally, it has been reported that blood flow in the ONH is autoregulated in response to fluctuations in systemic BP [3]. The difference in blood flow regulation in the retina and choroid may be related to the stronger autoregulatory mechanism of blood flow in the retina [2]. In the present study, the retinal blood flow velocity in the ONH increased immediately after the cold pressor test, when the activity of the sympathetic nervous system was high; accordingly, the BP and OPP also increased. These results indicate that sympathetic hyperactivity attributable to cold stimulation changes systemic circulatory hemodynamics but does not affect the retinal vessels.
Numerous studies have investigated the association between ONH and MBR in diseases that cause retinal circulatory disturbances. In the acute phase of central RVO, a pathology involving retinal circulatory disturbance, MBR-V, MBR-T, and MBR-A of ONH are significantly decreased [31]. Furthermore, abnormal ocular blood flow has recently been hypothesized as an important factor in visual field defects in glaucoma. A meta-analysis evaluating the diagnostic value of LSFG in glaucoma suggests that glaucoma is closely related to changes in ONH blood flow, as MBR-A, MBR-T, and MBR-V are reduced in glaucomatous eyes [32].
The present study has some limitations. First, the circulatory dynamics of the ONH were evaluated only through LSFG; however, OCT angiography should also be used for a detailed evaluation. Furthermore, although only the circulatory dynamics of the ONH were determined in this study, they should be measured using OCT C-scan to examine the relationship with the morphology of the ONH. Therefore, further studies are warranted to explore hemodynamic and morphological changes in the ONH after cold pressor test using different modalities. Second, BP varies with time of day, age, sex, season, stress, hormones, etc., and should be studied in detail under different conditions. Third, we performed the hand cold pressor test as the stimulation method in the present study; however, to enhance the applicability of our findings, it is necessary to measure under various stimulation conditions, encompassing whole body and foot cold pressor tests, as well as exercise stress tests.
In conclusion, sympathetic hyperactivity induced by the cold pressor test increased systemic circulatory dynamics, but not retinal circulatory hemodynamics, of healthy individuals, thus suggesting the involvement of vascular autoregulation. In the future, the findings from this study may provide basic data for future investigations of the relationship between diseases involving these retinal circulation disorders and the autoregulation of the ONH.
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