Autoregulation of Retinal Blood Flow in Response to Decreased Ocular Perfusion Pressure in Cats: Comparison of the Effects of Increased Intraocular Pressure and Systemic Hypotension

Abstract

Purpose.: To investigate the regulatory mechanisms responsible for autoregulation of retinal blood flow (RBF) during periods of decreased ocular perfusion pressure (OPP).

Methods.: The effects of acute reductions in OPP on RBF were assessed using laser Doppler velocimetry in cats. The OPP decreased from 90 to 40 mm Hg by increasing the IOP (elevated IOP) or by decreasing the systemic blood pressure via exsanguination (systemic hypotension). The contributions of nitric oxide (NO), adenosine, and/or N-methyl-D-aspartic acid (NMDA) in regulation of the retinal arteriolar hemodynamics during decreased OPP was determined at 120 minutes after intravitreal injection of various inhibitors or PBS.

Results.: Following PBS injection, the flow velocity decreased in proportion to the decrease in OPP; however, the retinal arteriolar diameter gradually increased. Consequently, the RBF was maintained near baseline levels when the OPP exceeded 70 mm Hg but decreased significantly (P < 0.01) when the OPP fell to less than or equal to 60 mm Hg due to elevated IOP or systemic hypotension. Adenosine receptor blocker 8-(p-sulfophenyl)theophylline, significantly (P < 0.01) enhanced decreases in RBF induced by elevated IOP and systemic hypotension at OPP from 80 to 40 mm Hg, whereas NO synthase inhibitor N ^G-nitro-L-arginine-methyl ester and NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid only significantly (P < 0.01) enhanced reductions in RBF induced by elevated IOP.

Conclusions.: These results indicate that adenosine contributes to autoregulation of RBF during systemic hypotension, whereas adenosine, NO, and NMDA receptors autoregulate the RBF after elevated IOP. Different vasoregulatory factors might contribute to autoregulation of RBF after decreases in OPP induced by elevated IOP and systemic hypotension.

Introduction

The autoregulation of blood flow, which is defined as the intrinsic ability of tissue to maintain relatively constant blood flow despite variations in perfusion pressure, ¹ operates in the coronary, ² renal, ³ cerebral, ⁴ and retinal microcirculation. ⁵ Ocular perfusion pressure (OPP) (i.e., the mean arterial blood pressure [MABP] minus IOP ⁶ ) is affected by changes in systemic blood pressure (BP) and IOP. Most of the previous studies that examined the changes in retinal blood flow (RBF) induced by acute decreases in OPP did so by increasing the IOP. ^7–9 However, increases in IOP are considered to lead to deformation of the lamina cribrosa, ¹⁰ which results in damage to the axons within the optic nerve and might also cause the direct compression of retinal ganglion cells (RGCs) and axons, and disturbances in axonal transport. ¹¹ Therefore, elevated IOP might cause hypoperfusion and place mechanical stress on retinal neuronal tissue. In contrast, decreases in the systemic BP are thought to cause only hypoperfusion in the retinal vessels (i.e., no mechanical stress is exerted on the retinal neuronal tissue). However, few experimental studies have investigated the dynamic changes in RBF that occur in response to acute decreases in OPP induced by reductions in systemic BP. 

When the decrease in the OPP exceeds the lower limit of RBF autoregulation, some metabolic factors (i.e., adenosine or glutamate) are produced by the surrounding neuronal tissue to compensate for the resultant tissue hypoxia caused by the decreased blood flow. Although adenosine ¹² and N-methyl-D-aspartic acid (NMDA) ¹³ induce vasodilation of retinal arterioles, it is unclear whether these metabolic factors are involved in the response of the RBF to reductions in OPP. 

In addition, a previous animal study reported that nitric oxide (NO) contributes to the regulation of the RBF in response to elevated IOP in newborn piglets. ⁹ However, the investigators administered NO synthase (NOS) inhibitors intravenously that increased the systemic BP and perfusion pressure. In our previous study, we injected an NOS inhibitor (N ^G-nitro-L-arginine-methyl ester [L-NAME]) into the vitreous to minimize the systemic effects and confirmed that the systemic BP did not change after the intravitreal injection. ¹⁴ In addition, based on experiments using laser Doppler velocimetry (LDV) in anesthetized cats, we showed that NO plays an important role in the regulation of RBF in response to systemic hypoxia, ¹⁴ hypercapnia, ¹⁵ hyperoxia, ¹⁶ and hypertension. ¹⁷  

The primary goal of the current study was to investigate the changes in RBF that occur in response to the decreases in OPP induced by elevated IOP or systemic hypotension. The second goal was to investigate whether NO, adenosine, and/or the glutamate receptor are involved in the changes in RBF that occur in response to reductions in OPP. 

Materials and Methods

Animal Preparation

The Animal Care Committee of Asahikawa Medical College approved the protocols involving the use of cats, and the study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Fifty-eight adult cats of either sex were used (weight, 2.2–3.6 kg). Anesthesia was induced with sevoflurane, oxygen, and nitrous oxide in a closed box followed by an intraperitoneal injection of atropine (0.04 mg/kg). The animals were tracheostomized and mechanically ventilated with 1.5% to 2.0% sevoflurane and room air. We placed catheters in the femoral arteries and the left femoral vein. Pancuronium bromide (0.1 mg/kg/h; Daiichi Sankyo Co., Tokyo, Japan) was infused continuously. A blood gas analyzer (Model ABL5; Radiometer, Copenhagen, Denmark) was used to intermittently measure the arterial pH, arterial partial carbon dioxide tension, and arterial partial oxygen tension. The MABP and heart rate were monitored continuously. A heated blanket was used to maintain each cat's rectal temperature between 37°C and 38°C. The pupils were dilated with 0.5% tropicamide (Santen Pharmaceutical Co., Osaka, Japan); a 0-diopter contact lens was placed on the cornea, and a drop of sodium hyaluronate (Healon; Abbott Medical Optics, Inc., Santa Ana, CA) was instilled. A 26-gauge butterfly needle connected to a bottle of balanced saline solution (BSS Plus; Alcon Laboratories, Fort Worth, TX) was inserted into the anterior chamber and connected to a pressure transducer. The IOP was monitored continuously. 

RBF Measurement

We used an LDV system (Laser Blood Flowmeter, model 100; Canon, Inc., Tokyo, Japan) customized for feline use to measure the RBF. The instrument simultaneously measures blood vessel diameter and blood flow velocity in the retinal vessels and automatically calculates the RBF, as described previously. ¹⁴ Laser Doppler velocimetry measurements were obtained from a retinal artery in one eye of each animal. The first-order arterioles chosen for measurement had relatively straight segments that were sufficiently distant from the adjacent vessels. 

The RBF was calculated according to the formula RBF = S × V _mean, where S is the cross-sectional area of the retinal artery at the LDV measurement site, assuming a circular cross-section, and V _mean is the mean blood velocity calculated as V _mean = V _max/2. ¹⁸ The MABP was determined using the formula MABP = diastolic BP + (systolic BP − diastolic BP)/3. Because the cats were in the prone position during the experiments, the OPP was calculated using the formula OPP = MABP − IOP. ⁶ Retinal arterial vascular resistance (RVR) was determined using the formula RVR = OPP/RBF. ¹⁴  

Intravitreal Injections

We administered intravitreal microinjections by inserting a 30-gauge needle attached to a 100-μL syringe (Hamilton, Reno, NV) into the vitreous at 3-mm posterior to the limbus. The tip of the needle was positioned over the optic disc. 

The involvement of NO, adenosine, or the glutamine NMDA receptor in the responses to the reductions in OPP elicited by elevated IOP and systemic hypotension was assessed in the presence of known effective concentrations of the following inhibitors: L-NAME (100 mM), a nonselective inhibitor of NO synthesis ^14,19 ; 8-(p-sulfophenyl)theophylline hydrate (8-SPT) (15 mM), an adenosine receptor blocker ²⁰ ; and DL-2-amino-5-phosphonopentanoic acid (DL-APV) (5 mM), an NMDA receptor antagonist. ²¹ Phosphate-buffered saline was the vehicle for all the drugs. Phosphate-buffered saline or one of the previously mentioned pharmacological inhibitors was administered intravitreally in a total volume of 50 μL. 

Experimental Protocols

The RBF measurements started 5 minutes before the injection and continued every 15 minutes for 120 minutes after the injection. The mean of five measurements obtained at 1-minute intervals before the injection was defined as the baseline RBF value. At each time point, we obtained three successive measurements and recorded the mean of the three measurements. 

The reductions in the OPP were induced 120 minutes after the intravitreal injection of PBS or an inhibitor. The RBF was measured at 10-mm Hg intervals (80, 70, 60, 50, and 40 mm Hg). Two separate measurement protocols were used to decrease the OPP: one by varying the IOP at a fixed MABP of 100 mm Hg (elevated IOP) and the second by varying the MABP at a fixed IOP of approximately 10 mm Hg (systemic hypotension). 

In the first protocol, the IOP level was increased in 10-mm Hg stepwise increments by placing a bottle connected to the anterior chamber at the corresponding height. It was maintained at each level for 2 minutes. The MABP was maintained at approximately 100 mm Hg throughout the experiment. The RBF was measured every 30 seconds from 60 to 120 seconds at each OPP level, and the mean of the three measurements was recorded. 

In the second protocol, the MABP was reduced gradually over 5 minutes via continuous withdrawal of blood from the descending aorta using a syringe pump until 24% of the total blood volume had been removed (Model 11 Plus Advanced; Harvard Apparatus, Holliston, MA), and the IOP level was maintained at approximately 10 mm Hg throughout the experiment. The RBF was measured at OPPs of 80, 70, 60, 50, and 40 mm Hg, and the mean of two measurements was recorded. 

Measurement of Vitreous Oxygen Tension Near the Retina

In another series of experiments, we measured the vitreous oxygen tension (P_vrO₂) close to the retina, which reflects the retinal oxygen tension (PO₂), as described previously. ²² Oxygen microelectrodes (POE-10N; BRC, Inc., Nagoya, Japan) were introduced into the optically intact eye through a 30-gauge needle inserted approximately 3-mm posterior to the limbus. The needle was held in a manipulator, which allowed the electrodes to be positioned close to the site at which the RBF in the retinal arterioles was measured by the LDV system. 

Chemicals

N ^G-nitro-L-arginine-methyl ester, 8-SPT, and DL-APV were dissolved in PBS. All chemicals were purchased from Sigma Chemical Co., St. Louis, MO. 

Statistical Analysis

All data are expressed as the mean ± SEM. The Student's paired t-test was used to compare the systemic and retinal parameters obtained before and after the change in OPP and those obtained before and after the intravitreal injections. One-way repeated-measures ANOVA followed by Dunnett's procedure was used to analyze the changes in the retinal circulatory parameters at different OPP levels. Two-way ANOVA followed by the Bonferroni procedure was used to compare the results of the L-NAME, 8-SPT, DL-APV, and vehicle groups. P less than 0.05 was considered significant. 

Results

Effects of Intravitreal Injections on the Retinal Circulation

There were no significant differences in any retinal parameters before injection among the different injection groups (Table 1). At 120 minutes after the intravitreal injection of PBS (n = 15), L-NAME (n = 11), 8-SPT (n = 12), or DL-APV (n = 12), no feline systemic hemodynamic parameters were altered significantly (data not shown). The intravitreal injection of PBS, 8-SPT, or DL-APV did not affect any of the retinal circulatory parameters examined; however, L-NAME induced decreases in retinal arteriolar diameter, blood flow velocity, and RBF compared with the baseline values (Table 1). 

Table 1

 

Effect of Intravitreal Injections on Retinal Circulation During Rest

	PBS, n = 15		L-NAME, n = 11		8-SPT, n = 12		DL-APV, n = 12
	Before	120 min	Before	120 min	Before	120 min	Before	120 min
Diameter, μm	98.1 ± 1.8	98.3 ± 3.8	104.6 ± 1.5	95.5 ± 1.2*	98.1 ± 1.6	98.3 ± 2.2	97.6 ± 2.0	97.8 ± 2.6
Velocity, mm/s	31.4 ± 3.1	32.7 ± 8.3	36.0 ± 1.3	26.1 ± 1.7*	31.6 ± 3.3	32.1 ± 3.8	31.5 ± 3.1	32.1 ± 3.7
RBF, μL/min	7.4 ± 0.9	7.7 ± 2.7	9.4 ± 0.4	5.5 ± 0.4*	7.4 ± 1.1	7.7 ± 1.3	7.3 ± 1.1	7.6 ± 1.3
RVR, mm Hg min/μL	14.8 ± 1.9	14.7 ± 3.9	10.1 ± 0.7	16.5 ± 1.1*	14.6 ± 1.7	14.2 ± 1.8	14.5 ± 1.6	14.3 ± 1.8

 Data are expressed as the mean ± standard error. Before, before the intravitreal injections; 120 min, 120 minutes after the intravitreal injections.

 * P < 0.05 for the comparison of the baseline and postinjection values using the paired Student's t-test.

Systemic Parameters Before and After OPP Reduction

Because MABP and IOP were maintained at approximately 100 and 10 mm Hg, respectively, the OPP was approximately 90 mm Hg (baseline; mean ± SD, 90.4 ± 2.5 mm Hg; range, 85.3–95.0 mm Hg) before the IOP was elevated or systemic hypotension induced. 

In the group with elevated IOP in which the OPP was decreased with increases in the IOP (n = 26), the IOP level increased significantly (P < 0.01) from a mean of 9.5 ± 2.0 to 58.5 ± 4.7 mm Hg, but the systemic BP did not change. In contrast, in the systemic hypotension group (n = 24), the MABP decreased significantly (P < 0.01) from a mean of 101.6 ± 4.9 to 51.3 ± 2.1 mm Hg and the IOP level remained unchanged. In all groups in which the systemic hypotension was changed, the arterial pH increased slightly, but significantly (P < 0.01), by the end of the experiment (Table 2). 

Table 2

 

Systemic Parameters Before and After Decreases in OPP

	Elevated IOP
	PBS, n = 8		L-NAME, n = 6		8-SPT, n = 6		DL-APV, n = 6
	Before	After	Before	After	Before	After	Before	After
MABP, mm Hg	98.5 ± 1.0	97.3 ± 1.9	100.2 ± 3.8	95.7 ± 2.3	100.6 ± 1.3	99.7 ± 1.6	100.4 ± 0.2	101.2 ± 1.2
IOP, mm Hg	9.3 ± 0.8	57.1 ± 1.8*	9.7 ± 1.1	56.0 ± 2.2*	9.4 ± 0.9	60.0 ± 1.0*	9.7 ± 0.2	61.5 ± 1.6*
OPP, mm Hg	89.3 ± 1.0	40.1 ± 0.4*	90.4 ± 2.6	39.7 ± 1.1*	91.0 ± 0.7	39.7 ± 0.7*	90.6 ± 0.2	39.8 ± 0.4*
HR, beats/min	140.8 ± 4.3	141.9 ± 4.3	150.8 ± 4.9	150.0 ± 5.3	151.5 ± 6.2	149.1 ± 6.8	156.0 ± 4.7	155.7 ± 5.0
pH	7.38 ± 0.01	7.42 ± 0.01	7.39 ± 0.01	7.37 ± 0.01	7.39 ± 0.01	7.40 ± 0.02	7.40 ± 0.02	7.39 ± 0.01
PaCO2, mm Hg	29.1 ± 0.6	30.4 ± 0.3	29.3 ± 0.9	29.0 ± 0.4	30.3 ± 1.0	30.0 ± 0.4	30.3 ± 1.3	31.2 ± 1.2
PaO2, mm Hg	112.5 ± 1.7	115.4 ± 3.7	110.0 ± 1.1	111.2 ± 2.3	114.2 ± 1.1	116.0 ± 1.7	116.2 ± 1.8	114.0 ± 2.8
HCO3 −, mEq/L	19.6 ± 1.1	20.9 ± 1.2	19.5 ± 0.2	20.8 ± 0.6	19.3 ± 0.2	20.5 ± 0.7	20.6 ± 0.2	20.8 ± 0.6

 Data are expressed as the mean ± standard error. Before, before decreases in OPP; After, after decreases in OPP; HR, heart rate.

*    P < 0.05 for the comparison of the baseline and postinjection values using the Student's paired t-test.

Table 2

 

Extended

	Systemic Hypotension
	PBS, n = 7		L-NAME, n = 5		8-SPT, n = 6		DL-APV, n = 6
	Before	After	Before	After	Before	After	Before	After
MABP, mm Hg	99.5 ± 2.7	52.1 ± 0.8*	104.8 ± 2.2	51.5 ± 1.4*	101.5 ± 1.1	50.0 ± 0.7*	101.1 ± 0.5	51.4 ± 0.3*
IOP, mm Hg	11.3 ± 0.8	11.4 ± 0.9	11.8 ± 0.8	12.2 ± 1.3	11.2 ± 0.4	10.2 ± 0.3	10.6 ± 0.2	10.1 ± 0.3
OPP, mm Hg	88.1 ± 2.2	40.7 ± 0.7*	93.0 ± 1.9	39.4 ± 0.4*	90.3 ± 0.9	39.8 ± 0.7*	90.5 ± 1.4	41.3 ± 0.1*
HR, beats/min	147.0 ± 6.7	153.6 ± 7.4	146.5 ± 5.3	149.3 ± 8.6	155.3 ± 5.6	155 ± 3.8	156.5 ± 3.7	163.5 ± 4.2
pH	7.42 ± 0.01	7.50 ± 0.02*	7.43 ± 0.01	7.48 ± 0.01*	7.41 ± 0.01	7.50 ± 0.02*	7.41 ± 0.02	7.47 ± 0.02*
PaCO2, mm Hg	32.2 ± 1.5	32.0 ± 1.0	30.5 ± 1.8	29.7 ± 1.3	32.8 ± 1.3	29.7 ± 1.0	31.8 ± 1.6	30.0 ± 1.7
PaO2, mm Hg	111.6 ± 2.7	117.0 ± 4.2	114.0 ± 4.8	119.5 ± 8.5	113.7 ± 4.8	115.7 ± 4.1	119.0 ± 2.4	121.4 ± 2.5
HCO3 −, mEq/L	19.3 ± 1.4	21.1 ± 0.9	17.9 ± 2.0	18.4 ± 2.4	18.6 ± 1.4	21.1 ± 1.1	18.7 ± 1.6	20.6 ± 0.9

Changes in Retinal Hemodynamics and Retinal Oxygen Tension After IOP Elevation or Systemic Hypotension

In the group in which the IOP was elevated in response to PBS, the blood velocity significantly (P < 0.01) decreased at an OPP of 80 mm Hg compared with the baseline (OPP of 90 mm Hg) and decreased by 44.9 ± 18.6% of its baseline value at an OPP of 40 mm Hg (Fig. 1). In contrast, the retinal arteriolar diameter gradually increased and reached significance (P < 0.01) at an OPP of 60 mm Hg. As a result, the RBF was maintained at a level that was similar to that at baseline when the OPP level exceeded 70 mm Hg but significantly (P < 0.01) decreased compared with the baseline value when the OPP decreased to 60 mm Hg or less (Fig. 1). Similarly, RVR also decreased significantly (P < 0.01) when the OPP decreased to 60 mm Hg or less. 

Figure 1

 

Retinal hemodynamics in response to reductions in OPP induced by elevated IOP or systemic hypotension following injection with PBS (n = 8 and 7, respectively). All retinal circulatory parameters are expressed as percentages of the baseline levels. *P < 0.05 and †P < 0.05 for comparisons with the baseline after the IOP was elevated or the systemic hypotension induced, respectively. D, diameter; V, velocity.

In the group in which systemic hypotension developed in response to PBS, the changes in all retinal circulatory parameters were comparable with those observed in the group in which the IOP increased in response to PBS (Fig. 1). There were no significant differences in the changes in retinal circulatory parameters induced in response to the reduction of the OPP level between the groups with elevated IOP and systemic hypotension. 

In another series of experiments, we measured the vitreous oxygen tension (P_vrO₂) in eight cats. In response to systemic hypotension (n = 4), the P_vrO₂ remained near the baseline values when the OPP exceeded 70 mm Hg and significantly decreased compared with the baseline values when the OPP was 60 mm Hg. When the OPP was 40 mm Hg, the P_vrO₂ decreased by 19.6 ± 8.7% of its baseline value. In response to elevated IOP (n = 4), the P_vrO₂ remained similar to the baseline levels when the OPP exceeded 60 mm Hg, but decreased significantly (P < 0.05) when the OPP was 50 mm Hg and decreased by 4.6 ± 4.1% of its baseline value when the OPP was 40 mm Hg (Fig. 2). The decreases in the P_vrO₂ induced in response to reductions in the OPP level differed significantly between the groups with elevated IOP and systemic hypotension. 

Figure 2

 

The P_vrO2 near the retina in response to reductions in OPP due to elevated IOP (n = 4) or systemic hypotension (n = 4). *P < 0.05 for comparisons with the baseline. †P < 0.05 for comparisons between the elevated IOP and systemic hypotension groups.

Effects of L-NAME, 8-SPT, and DL-APV on the Changes in RBF in Response to Reductions in the OPP Level

Pretreatment with L-NAME, 8-SPT, or DL-APV significantly (P < 0.05) enhanced the decrease in the RBF induced by elevated IOP compared with that seen in the group treated with PBS (Fig. 3). There were significant (P < 0.01) differences in the decrease in RBF induced in response to elevated IOP between the L-NAME group and the 8-SPT and DL-APV groups at OPP less than or equal to 60 mm Hg (Fig. 3). 

Figure 3

 

The changes in RBF induced by reductions in OPP caused by elevated IOP in the PBS (n = 8), L-NAME (n = 6), 8-SPT (n = 6), and DL-APV groups (n = 6). *P < 0.05 versus PBS group and †P < 0.05 versus L-NAME group.

Pretreatment with 8-SPT significantly (P < 0.01) enhanced the decrease in the RBF induced by systemic hypotension compared with that in the PBS group, whereas there was no significant difference between the PBS group and the L-NAME or DL-APV group in the decrease in RBF induced by systemic hypotension (Fig. 4). 

Figure 4

 

The changes in RBF induced by reductions in OPP caused by systemic hypotension in the PBS (n = 7), L-NAME (n = 5), 8-SPT (n = 6), and DL-APV groups (n = 6). *P < 0.05 versus PBS group.

Discussion

The current study showed that the blood velocity decreased significantly when the OPP level decreased to 80 mm Hg as a result of an increase in the IOP or a decrease in the systemic BP; however, the RBF remained unchanged when the OPP exceeded 70 mm Hg because of compensatory dilation of retinal arterioles (Fig. 1). These findings indicated that dilation of retinal arterioles plays an important role in the autoregulation of RBF in response to reductions in OPP. 

The current data also showed that the lower limit of RBF autoregulation lies between an OPP of 60 and 70 mm Hg in anesthetized cats (Fig. 1). It was previously reported that OPP values of 29.4 to 42 mm Hg represented the lower limit of RBF autoregulation after IOP elevations in humans ⁸ and animals. ^7,9 Although the lower limit of RBF autoregulation observed after the IOP was increased in the current study was higher than those reported previously, ^7–9,23 these discrepancies might have resulted from differences in the species examined, the experimental setups including the baseline OPP levels, and the techniques used to measure the RBF. 

In addition, no previous study has investigated the response of the blood flow in the retinal arterioles to reductions in the OPP elicited by systemic hypotension. In the current study, after inducing systemic hypotension by exsanguination, we showed for the first time that both RBF and P_vrO₂ were maintained at levels that were similar to the baseline values when the OPP exceeded 70 mm Hg (MABP = 80 mm Hg), but significantly decreased compared with the baseline level when the OPP was 60 mm Hg (MABP = 70 mm Hg). To investigate whether changes in RBF can prevent retinal tissue hypoxia in response to reductions in OPP induced by elevated IOP or systemic hypotension, we measured the oxygen tension in the vitreous humor (P_vrO2) close to the retina using oxygen microelectrodes. We confirmed that the P_vrO2 decreased with decreasing OPP during systemic hypotension but not during elevated IOP over the same range of the decrease in OPP (Fig. 2). Previous studies reported that PO₂ decreased during systemic hypotension in rats ²⁴ but not in cats with elevated IOP. ^25,26 These findings seem to agree with ours. The current results indicated that the RBF was regulated to maintain the retinal oxygen distribution in response to elevated IOP as a result of “autoregulation of RBF,” but this compensation of the RBF might be insufficient to prevent retinal tissue hypoxia in response to systemic hypotension. These discrepancies in the changes in retinal tissue oxygen tension in these two perturbations may be related to different vasoregulatory factors involved in autoregulation of the RBF in response to decreases in OPP in elevated IOP and systemic hypotension. 

Adenosine, a metabolite of cellular adenosine triphosphate (ATP), is a modulator of synaptic transmission and a potent endogenous vasodilator in most vascular beds including the retina. ^27,28 Interestingly, pretreatment with 8-SPT greatly enhanced the decrease in RBF induced by reductions in OPP in both the group with elevated IOP (Fig. 3) and that with systemic hypotension (Fig. 4), suggesting that adenosine is involved in the autoregulation of RBF in response to the decreases in OPP elicited by elevated IOP and systemic hypotension. Gidday and Park ¹² reported that 8-SPT attenuated the retinal vasodilator responses induced by systemic hypotension, which agrees with our findings. It was reported that retinal tissue hypoxia induced by ligation of the central retinal artery increased the extracellular concentration of adenosine in rat retinas. ²⁹ Although we could not measure the ocular concentration of adenosine in the current study, this finding seems to support our theory that adenosine is a key metabolite in the autoregulation of RBF in response to the decreases in OPP induced by elevated IOP and systemic hypotension. 

The current data indicated that autoregulation of the RBF in response to the reductions in OPP induced by elevated IOP was attenuated markedly by pretreatment with L-NAME (Fig. 3), whereas L-NAME did not affect the autoregulation of the RBF during systemic hypotension compared with the group treated with PBS (Fig. 4). A previous study reported that blockade of NOS did not attenuate the autoregulatory vasodilation of the retinal arterioles in newborn pigs during systemic hypotension. ³⁰ Although that study measured only vessel diameter, the findings were comparable to ours. In contrast, Jacot et al. ⁹ reported that L-NAME enhanced the decrease in regional RBF measured by radiolabelled microspheres induced in response to a reduction in OPP caused by elevated IOP, which also seems to support our results (Fig. 3). Taken together, our findings suggested that NO contributes to the autoregulation of RBF in response to reductions in OPP induced by elevated IOP but not those induced by systemic hypotension. 

Acute increases in IOP can cause neural degeneration via impaired glutamate metabolism. ³¹ We found that the NMDA receptor antagonist DL-APV enhanced the decrease in RBF induced by elevated IOP (Fig. 3) but not that induced by systemic hypotension (Fig. 4). This is to be expected because photoreceptors, bipolar cells, and ganglion cells are immunopositive for glutamate in cats. ³² Indeed, the intraretinal levels of glutamate increased during the first 10 minutes after the initiation of elevated IOP-induced ischemia. ³³ Moreover, isolated porcine retinal arterioles dilated in response to NMDA. ^13,28,34 Hence, it appears that activation of NMDA receptors via the increased glutamate levels in the retina might be involved in autoregulating RBF during IOP elevations. 

In the current study, we observed a significant difference in the extent of the decreases in RBF during IOP elevations observed between the L-NAME group and the 8-SPT or DL-APV groups (Fig. 3). In a previous study, acutely elevated IOP resulted in increased intraretinal levels of glutamate and subsequent abnormal activation of NMDA and non-NMDA glutamate receptor subtypes and increased NOS activity in rats, ³³ which seemed to agree with our results. A previous in vitro study found that the vasodilator effect of NMDA on porcine retinal arterioles was mediated by the hydrolysis of ATP to adenosine in perivascular retinal tissue ³⁴ and the vasodilation of isolated retinal arterioles to adenosine (≤10 μM) was demonstrated to be mediated in part by NO production, ^24,32 suggesting that NO is involved in the adenosine- and NMDA-induced vasodilation of retinal arterioles. Taken together, the current findings indicated that NO plays an important role in the preserving RBF in response to acute increases in IOP compared with adenosine and NMDA. Although we did not confirm which cells produced NO, adenosine, and glutamate in the current study, our results indicated that a close relationship exists between neuronal/glial cells in the retina and RBF regulation. 

We also found that L-NAME did not alter but 8-SPT enhanced the decrease in the RBF with systemic hypotension (Fig. 4), suggesting that adenosine plays a major role in the autoregulation of RBF during systemic hypotension independent of NO. A previous in vitro study ²⁴ showed that the activation of K_ATP channels is the predominant mechanism contributing to the dilation of retinal arterioles to the highest concentration (100 μM) of adenosine. Although we did not measure the concentration of adenosine in the retina and blood in our study, it is likely that these discrepancies in the role of adenosine in the RBF autoregulation during the decrease in OPP between systemic hypotension and elevated IOP may be associated with the difference in concentrations of adenosine produced. 

The current study had several limitations. First, systemic hypotension induced by exsanguination affected certain systemic circulatory parameters (e.g., metabolic alkalosis developed) (Table 2). Although there were no significant differences in the changes in these systemic parameters among the groups, we cannot exclude the possibility that the metabolic alkalosis affected the results in the group in which systemic hypotension was induced. Second, although sympathetic innervation previously was reported to be absent from retinal vessels, ³⁵ systemic hypotension induced by hemorrhaging might activate a sympathetic response. ³⁶ In our preliminary study, we confirmed that unilateral ganglionectomy did not affect the changes in RBF induced in response to systemic hypotension and elevated IOP (data not shown). Hence, the activation of autonomic innervation during systemic hypotension probably had little effect on our current findings. Third, our findings do not exclude the possibility that acute decreases in systemic BP induced by hemorrhaging might have decreased the perfusion pressure in the afferent glomerular arterioles, which might have increased their plasma renin and catecholamine levels. ³⁷ It was reported that the plasma level of catecholamine was unchanged in cats that lost 25% of their total blood volume. ³⁸ Because we induced systemic hypotension by withdrawing 24% of the total blood volume, it appears that the plasma catecholamine level does not play a major role in the autoregulation of the RBF in response to systemic hypotension. Further studies are needed to examine whether changes in the RBF induced by systemic hypotension are associated with the plasma level of catecholamine. Finally, we intravitreally administered 50 μL of 100 mM L-NAME into the feline vitreous to estimate the final concentration of 2 × 10⁻³ M of L-NAME in the vitreous, which seems to exceed the typical administration in the eye because 10⁻⁴ M L-NAME has been used to examine the role of NO in the eye in many in vitro studies. ^39–41 Because we could not measure the final concentration of L-NAME in the retina in our in vivo model, we used this concentration of L-NAME to confirm that L-NAME blocked NOS in the retina in our experimental setup. 

In conclusion, the RBF was maintained after induction of elevated IOP or systemic hypotension in cats when the OPP exceeded 70 mm Hg. When the OPP was decreased due to systemic hypotension, adenosine was the main factor involved in the autoregulation of RBF; however, adenosine, NO, and NMDA receptors contributed to the autoregulation of RBF after the IOP was elevated. Based on our findings, we believe that different vasoregulatory factors are involved in the autoregulation of RBF in response to the reductions in OPP due to elevated IOP and systemic hypotension. 

Acknowledgments

The authors thank Travis W. Hein and Lynda Charters for their technical support and manuscript review. 

Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (C-18591904 and B-25293352 [TN]). 

Disclosure: T. Tani, None; T. Nagaoka, None; S. Nakabayashi, None; T. Yoshioka, None; A. Yoshida, None