The Effect of High- to Low-Altitude Adaptation on the Multifocal Electroretinogram

Abstract

purpose. To examine variations in retinal electrophysiology assessed by multifocal electroretinogram (mfERG) during acclimatization of native highlanders to normobaric normoxia at sea level.

methods. Eight healthy residents of the greater La Paz area in Bolivia (3600 m above sea level) were examined over 72 days after arriving in Copenhagen, Denmark (sea level). A control group of eight healthy lowlanders was used for comparison.

results. During the period of observation, hemoglobin decreased from 16.7 to 15.0 g/dL (P = 0.0031), erythrocytes decreased from 5.3 to 4.6 trillion cells/L (P = 0.0006), and hematocrit decreased from 49.4% to 42.2% (P = 0.0008). At baseline, day 2 after arrival, the amplitudes (N1, P1, and N2) of the mfERG were 43.1% to 59.9% higher in the highlanders than in the lowlanders (P < 0.017). During acclimatization, the mfERG amplitudes increased 16.9% to 20.4% (P < 0.028) to a level of 73.2% to 87.0% higher in the highlanders than in the lowlanders (P < 0.0008). The increase in numerical amplitudes was proportional to the decrease in erythrocyte concentration (P = 0.023, 0.053, and 0.12 for N1, P1, and N2, respectively).

conclusions. On arrival at sea level, the highlanders had markedly supernormal multifocal electroretinographic amplitudes that continued to increase during the 72-day period of observation where the highlanders’ hematocrit normalized. The results suggest that acclimatization after a change in altitude and hence in ambient oxygen tension involves intrinsic retinal mechanisms and that acclimatization was not complete by the end of the study.

The retina has a high metabolic activity, ¹ yet this neurosensory tissue is sparsely vascularized in humans and completely avascular in several mammalian species. ^{2 3} In animals with circulatory patterns similar to those of humans, the middle layers of the retina are hypoxic and may reach anoxia during dark adaptation. ⁴ Neuronal function in this challenging metabolic environment is likely to be sustained wholly or partially by anaerobic glycolysis, which is supported by plasma lactate increasing from the arterial to the venous side of the retinal circulation. ⁵ Changes in electroretinographic potentials may be influenced by metabolic changes in the retina. Our previous studies of acute changes in blood glucose and arterial oxygen suggest that glucose affects latency (implicit times) whereas oxygen affects amplitude of the multifocal electroretinogram. ^{6 7 8} This finding agrees with those of similar studies of hypoxemia in both humans and animals. ^{9 10 11} Comparable observations have been made on humans during high altitude or simulated hypobaric hypoxia. ^{12 13 14} In our previous study, we further demonstrated an unequal eccentric distribution of the electroretinographic response to acute hypoxia. ⁸  

Sudden physiological perturbations tend to elicit not only acute but also chronic responses, often of a counterregulatory nature. In contrast to the mentioned acute-response studies, we have been unable to find any previous study of the chronic response to hypoxia on retinal electrophysiology. Whereas the chronic effects on the retina of high altitude are poorly described, the chronic systemic effects of high altitude are better understood and include increased hematocrit secondary to increased circulating levels of erythropoietin (EPO). ^{15 16 17}  

The purpose of this study was to examine the long-term effects of changes in ambient oxygen supply on retinal electrophysiological function and to correlate these findings to variations in hematology. During their 10-week stay at sea level after abrupt translocation, we examined a group of native highlanders normally residing around 3600 m above sea level (65% of sea level oxygen tension). 

Materials and Methods

Subjects

The study included eight healthy subjects who were all native inhabitants of the Bolivian highland (3600 m above sea level) where they were permanent residents and eight healthy control subjects who were all native inhabitants of the island of Zealand in Denmark (<50 m above sea level). The Bolivians were of Amerindian, European, and Mestizo ethnicity whereas the Danish subjects were ethnic Northern Europeans. All subjects were in good systemic and ocular health, in good nutritional condition, and had a better than average socioeconomic background. No subject used any systemic or ocular medication. Subjects were examined at 2, 23, and 72 days after arrival by plane after a two-leg flight from La Paz to Copenhagen. The study participants returned to Bolivia shortly after the 72-day examination. Follow-up in Bolivia could not be arranged. The subjects remained in or around Copenhagen during the entire study period. The Danish lowlanders were examined at comparable time intervals. The Bolivian highlanders were also included in separate studies of ocular lens autofluorescence ¹⁸ and measurement of retinal vessel diameter changes. ¹⁹ Written and oral informed consent was obtained from the participants after full explanation of the nature and possible consequences of the study. The study was approved by the medical ethics committee of Copenhagen County and adhered to the tenets of the Declaration of Helsinki. 

Methods

Study parameters included multifocal electroretinography (mfERG) in the subject’s right eye after pupil dilation to a diameter of ≥7 mm with 10% phenylephrine hydrochloride and 1% tropicamide. After topical anesthesia with 0.4% oxybuprocaine hydrochloride, a Burian-Allen bipolar contact lens electrode (Veris IR Illuminating Electrode; EDI Inc., San Mateo, CA) with two built-in infrared light sources for fundus illumination was placed on the cornea of the test eye using 1% carboxymethylcellulose as a contact fluid. The fellow eye was occluded. A ground electrode was attached to the forehead. 

Visual stimuli were displayed on a 1.5-in. stimulator/fundus camera (Veris; EDI, Inc.), permitting optimal correction of refraction without changing the size of the stimulus elements and ensuring fixation by real-time infrared observer viewing of the fundus. 

An array of 103 eccentricity-scaled hexagons was displayed at a frame rate of 75 Hz. Responses were band-pass filtered outside 10 to 300 Hz, amplified at gain 105, and sampled every 0.833 ms. A standard m-sequence length was used with m = 15, resulting in a total recording time of 7.17 minutes, divided into eight short segments for patient comfort. If loss of fixation or an artifact was observed the affected segment was discarded and rerecorded. The luminance of white stimuli was 200 versus 2 cd/m² or less for black stimuli. The surround luminance was set to 50% of the bright test luminance (i.e., 100 cd/m²). The examination was made in ambient room lighting. Stimulus luminance was calibrated using an autocalibrator, and the stimulus grid was calibrated using a grid calibrator (Veris; EDI, Inc.). The recording protocol agreed with the International Society for Clinical Electrophysiology of Vision (ISCEV) guidelines for basic mfERG. ²⁰  

Two iterations of artifact rejection were applied to the raw data, and no spatial smoothing was performed. The first- and second-order kernels were derived and implicit times and amplitudes of the mfERG components N1 (first negative), P1 (first positive), and N2 (second negative) were measured. The N1 response amplitude was measured from the starting baseline to the base of the N1 trough, the P1 response amplitude was measured from the N1 trough to the P1 peak, and the N2 response amplitude was measured from the P1 peak to the N2 trough. 

Average responses were calculated for the foveal stimulus hexagon and the surrounding five foveocentric rings of stimulus hexagons extending to an eccentricity of 25° (Fig. 1) . 

Additional investigations included best corrected visual acuity, intraocular pressure, slit lamp biomicroscopy, ophthalmoscopy, arterial blood pressure manometry, and venous blood analyses for erythrocyte and hemoglobin concentrations, hematocrit, and erythropoietin. Peripheral venous blood values were normalized for the effect of sex by scaling to the values of males. ²¹ All subjects had best corrected visual acuity 20/20 or better in both eyes and no detectable ophthalmic disease (Table 1) . 

Statistical analyses were performed with commercial software (SAS 9.1 software for windows; SAS Institute Inc, Cary, NC). A paired t-test was used for comparison between baseline and the two follow-up visits within the same group. A two-sample t-test was used for comparison between the two different groups. Correlations between study parameters were analyzed with ANOVA, and in the case of a significant result, a post hoc analysis was made with standard t-tests. The level of statistical significance was set at P < 0.05. 

Results

The groups of Bolivian highlanders and the Danish lowlanders (controls) were roughly sex- and age-matched. The two groups were comparable in sex, age, intraocular pressure, mean arterial blood pressure, blood glucose, and visual acuity (Table 1) . At baseline, 2 days after translocation from the altitude of 3600 m to near sea level, mfERG in the eight highlanders was of higher amplitude but of comparable latency to the eight lowlanders (Table 2) . The highlanders’ summed mfERG amplitude was 43.1% larger than the lowlander reference for N1, 47.1% for P1, and 59.9% for N2 (P = 0.017, 0.0037, and 0.0067, respectively). During the observation period, the mfERG amplitude in the highlanders continued to increase to even more supernormal levels, and in total the amplitudes increased 20.4%, 17.3%, and 16.9% (P = 0.013, 0.012, and 0.028 for N1, P1, and N2, respectively; Table 2 , Fig. 2 ). The rate of amplitude increase was lower after day 23. On day 72, the differences between the two groups were increased to 73.2%, 76.0%, and 87.0% for the three amplitudes. (P = 0.0008, 0.0005, and 0.0002, respectively). Scaling the summed mfERG responses from day 2 (baseline) by the peak to trough (N1–P1) of the day-72 responses revealed nearly identical waveforms at the starting and ending points of observation (Fig. 3) . 

These observations were consistent throughout the stimulated field, when tested by eccentricity: The P1 amplitudes increased during the observation period by 2.1% (0.6 nV/deg²) at 0° to 1.2° eccentricity (ring 1), 18.4% (4.7 nV/deg²) at 1.2° to 5° eccentricity (ring 2), 20.1% (3.6 nV/deg²) at 5° to 9° eccentricity (ring 3), 14.7% (2.2 nV/deg²) at 9° to 13.5° eccentricity (ring 4), 19.5% (2.4 nV/deg²) at 13.5° to 19° eccentricity (ring 5), and 18.4% (2.3 nV/deg²) at 19° to 25° eccentricity (ring 6) but only reached significance for the peripheral rings after Bonferroni correction (Table 3 , Figs. 1 4 ). Similar consistency throughout the stimulated field when tested by eccentricity was seen for the N1 and N2 amplitudes also. The N1 amplitude increased by 14.2% (ring 1), 22.9% (ring 2), 22.9% (ring 3), 23,1% (ring 4), 25.0% (ring 5), and 21.3 (ring 6); and the N2 amplitude increased by 11.7% (ring 1), 35.3% (ring 2), 19.1% (ring 3), 10.9% (ring 4), 10.0% (ring 5), and 9.5% (ring 6) (data not shown). 

On day 23 major changes for the P1 amplitude were seen centrally with an increase of 10.0% (3.7 nV/deg2) at 0° to 1.2° eccentricity (ring 1) and 8.4% (1.9 nV/deg2) at 1.2° to 5° eccentricity (ring 2), whereas at 5.6° to 25° eccentricity (rings 3–6) the P1 amplitude increased only 0.0% to 7.6% (Table 3) . Again, the same tendency was observed for the N1 and N2 amplitudes. On day 23, the N1 amplitude was increased by 20.9% (ring 1), 27.5% (ring 2), and 2.0% to 14.6% more peripherally (rings 3–6) and the N2 amplitude was increased by 17.9% (ring 1), 12.2% (ring 2) and 2.1% to 17.0% more peripherally (rings 3–6). 

Implicit times were comparable between highlanders and lowlanders throughout the study, the only significant change being a 1.5% shortening of the P1 implicit time in highlanders between baseline and day 72 (P = 0.034; Table 2 ). No significant mfERG changes over time were seen in the control group of lowlanders (Table 2) . The second-order amplitudes and implicit times were comparable between highlanders and lowlanders throughout the visits (data not shown). 

Blood samples drawn on day 2 showed hemoglobin 16.7 ± 0.6 g/dL (reference in healthy lowland subjects 13.8–17.2 g/dL), ²⁰ erythrocyte count 5.3 ± 0.2 million cells/μL (reference 4.7–6.1 million cells/μL), hematocrit 49.4% ± 2.2% (reference, 40.7%–50.3%), and erythropoietin 5.8 ± 1.3 mU/mL (reference, 0–19 mU/mL). Sea level acclimatization of the highlanders was evident as marked reductions in hemoglobin (−10.6% on day 72, P = 0.0031), hematocrit (−14.6% on day 72, P = 0.0008), and erythrocyte concentration (−13.6% on day 72, P = 0.0008) during the study period, whereas erythropoietin fluctuated within normal limits with an increase from baseline (day 2) to day 23 (+54.3%, P = 0.014) and day 72 (Table 4) . 

The subjects in the present study did not demonstrate significant vasoconstriction or vasodilation during the period of observation (P = 0.075 for veins and P = 0.11 for arteries). The participants in the present study were a subgroup of a larger study; the vessel diameter findings of that study has been reported elsewhere. ¹⁹  

The increase in numerical mfERG amplitudes in highlanders was proportional to the decrease in erythrocyte concentration for N1 of the mfERG, with the same trend for P1 and N2 (P = 0.023, 0.053, 0.12 and Pearson’s r = 0.76, 0.70, and 0.59 for N1, P1, and N2 respectively, summed mfERG; Fig 5 ). There were no correlations between mfERG changes and arterial blood pressure, blood glucose, intraocular pressure, hemoglobin, hematocrit, erythropoietin, or changes in vessel diameters. 

Discussion

We studied acclimatization over 10 weeks after acute transition to sea level of a group of native highlanders living at 3600 m of altitude. During descent from high altitude to sea level, potent cardiopulmonary and hematologic compensatory responses occur. ¹⁶ The same hematologic responses were observed in our study, with a decrease in hemoglobin (10.6%, P = 0.0031), erythrocytes (13.6%, P = 0.0006), and hematocrit (14.6%, P = 0.0008). The low erythropoietin level on day 2 and the succeeding increase is explained by an initial decrease in erythropoietin before being seen by us. This has been observed in other studies. ¹⁶ During acclimatization we observed remarkably supernormal mfERG amplitudes. The mfERG changes were uniformly distributed over all three amplitudes (N1, P1, and N2) and with comparable distribution according to retinal eccentricity. The nearly identical mfERG waveforms throughout the study suggest that the effect of relative hyperoxia and acclimatization was the same on all components of the mfERG. This pattern differed markedly from earlier studies of electroretinographic responses to acute changes in oxygen supply. ^{8 9 10 11 12 13 14} Although the present study involved mfERGs, whereas key prior studies applied full-field electroretinography, the responses recorded by either method have been shown to have the same cellular origin. ²² Hence, a comparison between studies seems valid, at least in qualitative terms. Despite the gradual dismantling of hematologic characteristics of high-altitude adaptation during the study period, the mfERG amplitudes of the highlanders continued to increase up to the latest time of follow-up, day 72 after arrival at sea level. This result seems to firmly reject the suggestion that the supernormal ERG may have been a consequence of the higher circulatory capacity of people adapted to high altitude that is well known from athletics. Probably the supernormal electroretinographic function of the retina is evidence of adaptation of neuronal function to a different metabolic environment and hence, by analogy with muscle work, a different type of retinal neuronal fitness. Such long-term adaptational phenomena have not previously been demonstrated in the retina, but analogous findings have been seen in the brain. In chronic hypobaric hypoxia, the brain cortex metabolism of mice shifts toward lower aerobic and higher anaerobic enzymatic activity, ^{23 24} and it is reasonable to assume that opposite compensatory processes occur when going from chronic hypoxia to normoxia, which may explain our ERG findings. This finding is supported by reports of retinal function being partially maintained by glycolysis, which is upregulated during respiratory chain depression. ²⁵  

High altitude natives (Tibetan and Andean) have a lower mitochondrial volume in leg muscle tissue than do sea level natives, along with other physiological traits that compromise oxygen utilization. ²⁶ These differences are presumed to be genetic due to an evolutionary adaptation process. ^{26 27} It is unclear to what extent hereditary differences may explain the difference between lowlanders and highlanders in the present study, but the change over time is clear evidence of a pronounced and prolonged process of acclimatization that does not follow the observed normalization of hematologic parameters. 

Although a statistical correlation was found in the present study between the mfERG amplitude changes and the changes in red blood cell concentration, the present study did not identify a common mechanism. A similar correlation between mfERG changes and changes in hemoglobin or hematocrit was not found, perhaps because of the limited power of the study. Our results suggest that recent changes in altitude of residence significantly affect ERG amplitudes, a finding that may need to be taken into consideration in ERG studies where absolute amplitude is of interest, whereas studies of variation in amplitude between different locations in the same eye are less likely to be affected. If it is assumed that the ERG of highlanders who move to sea level will eventually achieve ERG characteristics comparable with those of lowlanders, our findings suggest that the time to achieve sea level normalization is much longer than the 72-day duration of this study. Ideally, future studies will compare adaptation to high altitude and adaptation to low altitude and should probably extend to duration of at least 1 year and should include baseline examinations before departure, including examinations of retinal structure. 

 Supported by the Center for Biomedical Optics and new Laser Systems under the Danish National Advanced Technology Foundation; the Lundbeck Foundation Center for Neurovascular Signaling; a Patient-Oriented Diabetes Research Career Award (8-2002-130) from the Juvenile Diabetes Research Foundation (ML); and the Velux Foundation.

 Submitted for publication November 25, 2008; revised January 23, 2009; accepted April 21, 2009.

 Disclosure: P.K. Kofoed, None; B. Sander, None; G. Zubieta-Calleja, None; L. Kessel, None; K. Klemp, None; M. Larsen, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

The concentric ring grouping analysis. Ring 1 (0–1.2°), fovea; ring 2 (1.2–5°), parafovea; rings 3 (5–9°) and 4 (9–13.5°), mid macula; and rings 5 (13.5–19°) and 6 (19–25°), peripheral macula superimposed on the array of 103 hexagonal stimulus fields.

Table 1.

 

Clinical Characteristics of the Study Populations

	Highlanders	Controls	P *
Sex (men/women)	5/3	5/3
Age (y)	40.0 (10.5)	34.4 (5.8)	0.14
Intraocular pressure (mmHg)	11.7 (2.8)	13.2 (1.6)	0.16
MAP (mmHg)	84.9 (11.1)	93.8 (9.3)	0.65
Blood glucose in mmol/L	6.1 (0.8)	6.2 (0.6)	0.50
Altitude (m)	3600 (0)	Sea level
Visual acuity (Snellen)	1.0–1.2	1.0–1.2

 Values are presented as the mean (SD). MAP, mean arterial blood pressure.

*   t-Test between the two groups.

Table 2.

 

Amplitudes and Implicit Times for the Summed Multifocal ERG in Highlanders during Sea Level Acclimatization

	Day 2			Day 23							Day 72
		Mean	95% CI	Mean	95% CI	Δ%	P	Mean	95% CI	Δ%	P
Highlanders
Amplitudes (nV/deg2)
N1	−9.0	−10.9–−7.0	−10.2	−12.2–−8.3	14.3	0.063	−10.8	−12.9–−8.6	20.4	0.013
P1	17.1	14.1–20.1	17.9	14.9–20.9	4.6	0.29	20.1	16.1–24.1	17.3	0.012
N2	−13.6	−16.7–−10.6	−15.1	−17.7–−12.4	10.3	0.27	−16.0	−19.0–−12.9	16.9	0.028
Implicit times (ms)
N1	15.7	14.8–16.5	16.0	15.3–16.6	1.9	0.71	15.4	14.6–16.2	−1.6	0.23
P1	28.9	27.8–30.1	29.2	28.3–30.0	0.8	0.84	28.5	27.4–29.6	−1.5	0.034
N2	43.6	42.2–45.0	44.0	43.2–44.8	1.0	0.84	43.2	42.2–44.3	−0.9	0.31
Controls
Amplitudes (nV/deg2)
N1	−6.3	−7.6–−4.9	−6.4	−7.9–−5.0	2.7	0.24	−6.2	−7.6–−4.9	−0.5	0.92
P1	11.6	9.4–13.9	11.9	9.5–14.3	2.5	0.51	11.4	9.2–13.6	−2.0	0.74
N2	−8.5	−10.8–−6.3	−9.1	−11.4–−6.8	6.6	0.073	−8.5	−10.4–−6.7	0.0	1.00
Implicit times (ms)
N1	15.7	15.3–16.2	15.8	15.3–16.4	0.8	0.57	15.6	15.1–16.1	−0.6	0.35
P1	29.0	28.2–29.7	28.8	28.0–29.5	−0.7	0.48	28.9	28.1–29.6	−0.4	0.35
N2	43.0	42.1–44.0	43.1	42.5–43.7	0.2	0.73	43.2	42.3–44.2	0.5	0.34

 Percent changes and probabilities of differences for days 23 and 72 compared with day 2 (baseline; paired t-test) in highlanders versus lowlanders (control). 95% CI, 95% confidence interval.

Figure 2.

 

mfERG recording in a healthy Bolivian subject on days 2 (left) and 72 (right) after translocation from 3600 m of altitude to sea level.

Figure 3.

 

Summed mfERG responses from the eight Bolivian subjects on days 2 (dark curve) and 72 (gray curve). The responses on day 2 are scaled by the peak to trough (N1–P1) of the day 72 responses. The waveforms from the 2 days are nearly identical, which strongly suggests a similar effect on all components of the mfERG.

Table 3.

 

Highlander First-Order P1 Amplitudes Averaged from Six Concentric Rings during Sea Level Acclimatization

Rings	1	2	3	4	5	6
Day 2
Mean (nV/deg2)	53.6	23.9	17.5	15.6	12.6	13.5
95% CI	44.9–62.4	19.1–28.7	14.0–20.9	12.7–18.5	10.4–14.8	11.2–15.8
Day 23
Mean (nV/deg2)	57.3	25.8	18.3	15.6	13.5	13.5
95% CI	47.2–67.5	19.5–32.1	15.1–21.5	13.1–18.2	11.7–15.4	11.5–15.6
Δ%	10.0	8.4	4.0	0.1	7.6	0.0
95% CI	−1.4–21.4	−1.2–18.0	−4.6–12.6	−4.8–5.0	−11.5–26.7	−6.8–5.5
P	0.075	0.075	0.30	0.95	0.37	0.58
Day 72
Mean (nV/deg2)	54.3	28.6	21.0	17.8	15.0	15.8
95% CI	36.8–71.8	21.0–36.1	16.6–25.4	14.7–20.9	12.4–17.6	13.7–18.0
Δ%	2.1	18.4	21.0	14.7	19.5	18.4
95% CI	−30.9–35.1	4.5–32.3	2.7–39.3	3.4–26.0	8.6–30.3	7.4–29.4
P	0.88	0.017	0.030	0.018	0.0039	0.0055

 Percent changes with 95% CI and probabilities (paired t-test) of differences for days 23 and 72 relative to day 2 (baseline). P < 0.0083 was considered significant after Bonferroni correction.

Figure 4.

 

The relative distribution of first-order P1 amplitudes averaged from the six rings of increasing eccentricity during the observation period. Day 2 (baseline) is set to 100%. Horizontal bar: average value. Lowlanders served as the control. The changes were equally distributed throughout the six rings. The same pattern was observed for the N1 and N2 amplitudes.

Table 4.

 

Hematological Changes in Highlanders during Sea Level Acclimatization

	Day 2	Day 23	Day 72
Hemoglobin
Mean (g/dL)	16.7	15.6	15.0
95% CI	16.2–17.3	15.1–16.2	14.1–15.8
Δ%		−6.6	−10.6
P		0.012	0.0031
Erythrocytes
Mean (million cells/μL)	5.3	5.0	4.6
95% CI	5.0–5.5	4.8–5.2	4.3–4.8
Δ%		−5.3	−13.6
P		0.086	0.0006
Hematocrit
Mean (%)	49.4	46.5	42.2
95% CI	47.2–51.6	44.5–48.6	39.8–44.6
Δ%		−5.8	−14.6
P		0.075	0.0008
Erythropoietin
Mean (mU/mL)	5.8	8.9	8.7
95% CI	4.5–7.1	7.6–10.2	6.8–10.7
Δ%		54.2	50.7
P		0.014	0.068

 Percent changes and P (paired t-test) of differences for days 23 and 72 compared with day 2 (baseline).

Figure 5.

 

The correlation between mfERG N1 amplitude change and the decrease in red blood cell concentration from days 2 to 72 (P = 0.023 and Pearson’s r = 0.76).

The authors thank Gustavo Zubieta-Castillo, Sr, and Poul-Erik Paulev for valuable comments and suggestions.