April 2011
Volume 52, Issue 5
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Physiology and Pharmacology  |   April 2011
Disruption of Gap Junctions May Be Involved in Impairment of Autoregulation in Optic Nerve Head Blood Flow of Diabetic Rabbits
Author Affiliations & Notes
  • Maho Shibata
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Hidehiro Oku
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Tetsuya Sugiyama
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Takatoshi Kobayashi
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Mami Tsujimoto
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Takashi Okuno
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Tsunehiko Ikeda
    From the Department of Ophthalmology, Osaka Medical College, Osaka, Japan.
  • Corresponding author: Hidehiro Oku, Department of Ophthalmology, Osaka Medical College, 2-7 Daigaku-cho Takatsuki Osaka, 569-8686 Japan; hidehirooku@aol.com
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2153-2159. doi:10.1167/iovs.10-6605
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      Maho Shibata, Hidehiro Oku, Tetsuya Sugiyama, Takatoshi Kobayashi, Mami Tsujimoto, Takashi Okuno, Tsunehiko Ikeda; Disruption of Gap Junctions May Be Involved in Impairment of Autoregulation in Optic Nerve Head Blood Flow of Diabetic Rabbits. Invest. Ophthalmol. Vis. Sci. 2011;52(5):2153-2159. doi: 10.1167/iovs.10-6605.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To determine whether an impairment of the autoregulatory mechanism of blood flow in the optic nerve head (ONH) is present in diabetic rabbits and whether the impairment results from the uncoupling of gap junctions.

Methods.: Experiments were performed on six alloxan-induced diabetic rabbits and six healthy control animals. In a test of the integrity of the autoregulatory mechanism, the intraocular pressure (IOP) was elevated from the 20-mm Hg baseline to 50 and then to 70 mm Hg. The capillary blood flow in the ONH was measured every 10 minutes by the laser speckle method, with simultaneous measurements of blood pressure. Ocular perfusion pressure (OPP) was calculated at each step, and the relationship between blood flow and OPP was analyzed. In addition, octanol, gap27 (gap junction uncouplers), or balanced saline solution was injected into the vitreous of healthy rabbits, with the balanced saline solution–injected eyes serving as the control. Changes in the ONH blood flow in response to the IOP elevation were determined in the same way.

Results.: Diabetic rabbits had a significant decrease in ONH blood flow when the OPP was reduced by an elevation of the IOP to 50 or to 70 mm Hg, whereas the ONH blood flow was well maintained in healthy rabbits. After injection of octanol (10.0 mM) or gap27 (10 μM), a reduction of OPP resulted in a significant decrease in ONH blood flow in the healthy rabbits.

Conclusions.: These results indicate that autoregulation is disrupted in diabetic animals, and uncoupling the gap junctions in healthy rabbits also disrupts the autoregulation.

The retinal vessels and the vessels within the optic nerve head (ONH) lack innervation, but the blood flow in these regions is still altered to meet the metabolic needs through an autoregulatory mechanism. 1,2 An important function of autoregulation is to maintain constant blood flow in these regions when changes in the ocular perfusion pressure (OPP) occur. 2 The mechanism for this autoregulation has not been precisely determined, but endothelial cells, which secrete vasoactive molecules that regulate the vascular tone, are most likely involved. 
The blood–retinal barrier is made up of tight junctions that block the leakage of serum and its component macromolecules into the retinal tissue, whereas gap junctions couple neurons, glial cells, and vascular cells to each other, thus controlling the flow of fluids and molecules among these cells. This functional coupling allows electrical currents and signaling molecules to pass between cells, and disruption of these junctions impairs the coupling (e.g., vasodilatation) responses. 3,4 It has also been shown that the end feet of astrocytes surrounding the vascular walls form gliovascular interfaces, where connexin 43 (Cx43), a gap junction protein, and purinergic receptors are highly expressed. 5 Because purinergic receptors are involved in blood flow regulation, it is important to determine whether the metabolic needs of retinal neurons are conveyed through gap junctions of the gliovascular interfaces. 
In diabetic eyes, communication through gap junctions and the expression of Cx43 in the retinal vessels are reduced. 6 8 Although autoregulation in the retinal circulation is impaired in diabetic eyes, 9 whether this is also true of ONH blood flow has not been clearly determined. 10 Thus, it would be of interest to determine the effects of gap junction uncouplers on the blood flow to the ONH. 
We hypothesized that the autoregulatory mechanism that controls blood flow in the ONH is also impaired in the eyes of diabetic rabbits. To test this hypothesis, we examined the effects of elevating the intraocular pressure (IOP) on the blood flow to the ONH in alloxan-induced diabetic rabbits by the laser speckle method. 11 We also determined the effects of gap junction uncouplers on the ONH blood flow. 
Materials and Methods
Animals
Young adult albino rabbits (2.6–2.8 kg) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan) and housed in an air-conditioned room at approximately 23°C in 60% humidity and placed on a 12-hour light–dark cycle. Only the right eye was used in all the experiments. The experimental protocol was approved by the Committee of Animal Use and Care of the Osaka Medical College, and all animals were managed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Chemicals
All chemicals, unless otherwise noted, were purchased from Sigma-Aldrich (St. Louis, MO). 
Induction of Diabetes
Diabetes was induced in the rabbits by an intravenous injection of 80 mg alloxan/kg through an ear vein. Because alloxan causes transient hypoglycemia due to the release of insulin from the damaged β-cells, 20 mL of a 20% glucose solution was injected intravenously 6 hours after the injection, to prevent death from acute hypoglycemia. The experiments were performed 8 weeks after the induction of diabetes. 
Analysis of ONH Blood Flow
The capillary blood flow in the ONH was measured with the laser speckle method, a noninvasive, two-dimensional measurement of tissue circulation. 11,12 Briefly, the ocular fundus was illuminated with an 808-nm diode laser and observed through a retinal camera by a customized image sensor (100 × 100 pixels) with a high scanning rate. The objective field of the rabbit ONH was 0.72 mm2, which was imaged on the sensor, where a speckle pattern appeared due to the random interference of the scattered light from the ocular fundus. The structure of the speckle pattern varied in time according to the movement of the blood cells in the vessels and the capillaries. With an increase in the blood velocity in the object, the frequency of the time-varying image speckles increases. Accordingly, the output image from the sensor begins to be blurred in the exposure time of each scanning, and the variation of the intensities sampled at each pixel point decreases. The normalized blur (NB) value was introduced to characterize this blur rate, defined as the ratio of mean intensity to the SD of the intensity values. The NB is expressed as an arbitrary unit, and is known to be a quantitative index of the blood flow velocity. Also, the changes in the NB values show good correlation with the changes in capillary blood flow in the ONH. 13,14  
The rabbits were placed in a holding box, and measurements were performed with the animals under local anesthesia with 0.4% oxybuprocaine (Santen, Osaka, Japan). To evaluate the changes in the ONH capillary blood flow, we dilated the pupils with 0.4% tropicamide, and an area (0.72 mm2) on the ONH where no vessels were visible was measured. The NB values were serially determined, and the NB at each time point was calculated as the average of five successive measurements. It required 0.18 second to record 98 scans to obtain one NB value. 
Elevation of IOP
The IOP was artificially elevated by increasing the height of a bottle of balanced saline solution (BSS Plus; Alcon Laboratories, Fort Worth, TX), which was connected to the vitreous cavity through an infusion cannula. The placement of the infusion cannula was performed with the rabbit under local anesthesia with topical lidocaine (4.0%). The level of IOP was first adjusted to 20 mm Hg for the baseline measurements and then increased to 50 and to 70 mm Hg by monitoring the levels with a pressure transducer (P10EZ; Gould Statham Instruments, Hatorey, Puerto Rico). 
Effects of Elevated IOP and OPP on ONH Blood Flow
Changes in the capillary blood flow in the ONH in response to elevated IOPs were measured by the laser speckle method in diabetic (n = 6) and healthy rabbits (n = 6). The baseline level of ONH blood flow was measured 10 minutes after the IOP was set to 20 mm Hg. The IOP was then set at 50 or 70 mm Hg, and measurements were made every 10 minutes at each IOP level for 30 minutes. Thus, in addition to the baseline blood flow, three measurements at each IOP level were recorded, for a total of seven measurements in each rabbit. 
The blood pressure was simultaneously recorded at each IOP level. The systemic arterial blood pressure (BP) and heart rate were measured on the front leg with an automatic sphygmomanometer (BP-98E; Softron, Tokyo, Japan). The accuracy of this method has been reported. 15 The relationship between ONH blood flow and OPP was analyzed by plotting the OPPs on the abscissa and the NB values relative to the baseline on the ordinate. 
The OPP was calculated by using the standard equation 16 :   where MBP = DBP + ⅓(SBP − DBP). MBP is the mean arterial blood pressure, DBP is the diastolic blood pressure, and SBP is the systolic blood pressure. 
Effect of Octanol and Gap27, Gap Junction Uncouplers
Gap junction communication was disrupted by an intravitreal injection of the gap junction uncouplers octanol 3,17,18 or gap27. 19,20 The octanol solution was diluted in balanced saline solution to 10-, 30-, and 100-mM concentrations, and gap27 was dissolved in the balanced saline solution to make a 100-μM solution. Octanol at each concentration or gap27 solution (200 μL each) was injected into the vitreous through the pars plana with a 30-gauge needle fitted to a syringe (Hamilton, Reno, NV), with the rabbit under local anesthesia with 0.4% oxybuprocaine (Santen). The concentration of octanol in the rabbit vitreous of approximately 2.0 mL was ∼1.0 (n = 6), 3.0 (n = 6) and 10.0 (n = 6) mM, respectively. The concentration of gap27 was 10 μM (n = 4) in the rabbit vitreous. 
These concentrations were chosen because octanol (1.0 mM) has been shown to disrupt intercellular communication in juxtaglomerular cells 18 and in rabbit retinal neurons. 21 It has also been demonstrated that gap27 at 300 μM impairs endothelium-derived vasodilatation. 22 However, 100 μM gap27 caused vitreous opacity that impaired the visibility of the ocular fundus of the rabbits. Thus, we used 10 μM gap27. In the controls, 200 μL of balanced saline solution was injected into the posterior vitreous (n = 10). Before the injections, the pupils were dilated with 0.4% tropicamide. In the end, 32 rabbits were used to analyze the effects of gap junction uncouplers; 18 received different concentrations of octanol, 4 were injected gap27, and 10 served as controls. 
Two hours were allowed for the chemicals to be absorbed into the vascular system of the ONH. Then, the infusion cannula was inserted into the vitreous cavity, and the IOP was increased from 20 (baseline) to 50 or 70 mm Hg. The examiner who measured the ONH blood flow was masked to prior treatments of the eye. 
Assessment of Retinal Function
To determine whether gap junction blockers alter the activity of retinal neurons, we recorded electroretinograms (ERGs) and visually evoked potentials (VEPs) before and 3 hours after the intravitreal injection of octanol (10 mM, n = 4) or gap27 (10 μM, n = 4). The ERGs were recorded with the pupil of the right eye fully dilated with 0.4% tropicamide and the eye held open with a Barraquer wire speculum. The left eye was completely covered to prevent stray-light stimulation. A diffuser was placed before the stimulated right eye to ensure full-field stimulation. The ERGs were elicited by 20 J light stimuli 20 cm in front of the eye, and recordings were made with a gold-ring active electrode that was attached to a contact lens placed on the cornea. The mean luminance at the corneal surface was 690 cd/m2, and the duration was 60 ms. Before the ERGs were recorded, the animals were dark adapted for 60 minutes, and four responses were averaged to the light stimuli (SLS 4100) at 0.1 Hz. The band-pass filters (AVM-10, Nihon-Kohden. Tokyo, Japan) were set at 1.5 and 100 Hz, to record the a- and b-waves, and at 50 and 300 Hz, to record the oscillatory potentials (OPs) of the ERGs. We measured implicit times (ITs) and amplitudes of a- and b- waves of the ERGs. 
Our technique for recording VEPs has been published in detail. 23 Briefly, at least 2 weeks before the experiments, the active and reference electrodes (M2–15; Unique Medical, Tokyo, Japan) were implanted on the dura in rabbits under general anesthesia with intraperitoneal urethane (0.8 g/kg). The VEPs elicited by stimulating the right eye were recorded from the active electrode placed over the left primary visual area, which was 6 mm anterior and 6 mm lateral to the lambda point. The animal was grounded by an electrode on the right ear. 
VEPs were elicited by 0.6 J photic stimuli from conscious but restrained animals. The signals were amplified and the band-pass filters were set at 1.5 to 100 Hz. A signal averager (DAT-1100; Nihon-Kohden) was used to summate 32 responses. The light source was placed 60 cm in front of the eye. The mean luminance at the corneal surface was 2.3 cd/m2, and the duration was 60 ms. 
The analog data were recorded on a rectilinear pen recorder, and the data were also fed in parallel to a computer, where they were digitized and stored for later analyses (MacLab 2e; AD Instruments, Castle Hill, Australia). The first negative peak (N1) is the most prominent one of the rabbit VEP and appeared at approximately 20 ms after the stimulus. The IT of the N1 peak was automatically calculated by the computer program for each VEP. The reliability of the recordings was determined earlier; the mean coefficient of variation of the IT of the N1 peak was 0.4%. 23  
Statistical Analyses
The data are expressed as the mean ± standard deviation (SD), unless otherwise noted. Statistical analyses were performed by one-way analysis of variance (ANOVA) or two-way, repeated-measures ANOVA. Student's t-tests were used to compare two groups. If the distribution of the data was different, the Welch correction was used for t-tests of samples with unequal variations. Interactions between ONH blood flow and OPP were analyzed by simple linear regression. The level of significance was set at P < 0.05. 
Results
ONH Blood Flow in Diabetic Animals
Six rabbits were successfully made diabetic 8 weeks after the injection of alloxan, with blood sugar levels of 468.0 ± 83.8 mg/dL in the alloxan-injected rabbits (n = 6) and 96.0 ± 18.5 mg/dL in the controls rabbits (n = 6). The average body weight was 2.7 ± 0.3 kg in the diabetic rabbits and 3.2 ± 0.3 kg in the controls. 
Alterations in the ONH blood flow expressed by the NB values relative to the baseline are shown in Figure 1. In the healthy animals, the ONH blood flow was maintained at 94.1% ± 9.4% and 91.5% ± 9.4% of the baseline when the IOP was elevated to 50 and 70 mm Hg, respectively. In contrast, the NB values were decreased to 73.8% ± 13.8% of the baseline when the IOP was raised to 50 mm Hg and to 51.6% ± 11.5% when the IOP was raised to 70 mm Hg in the diabetic animals. Two-way interactions showed that the differences were significant (P = 0.0002, repeated-measures ANOVA), and the differences between diabetic and control animals were significant at each assessment time point (P < 0.05, t-tests). 
Figure 1.
 
Changes in the ONH blood flow (or NB) relative to the baseline in response to IOP elevation. Data are expressed as the mean ± SD. A decrease in ONH blood flow after IOP elevation to 50 and 70 mm Hg was observed in the diabetic animals, whereas the blood flow was well maintained in the healthy animals. Two-way interaction is significant (P = 0.0002, repeated-measures ANOVA), and the differences between the two groups at each assessment time point are significant (*P < 0.05, **P < 0.01, t-tests).
Figure 1.
 
Changes in the ONH blood flow (or NB) relative to the baseline in response to IOP elevation. Data are expressed as the mean ± SD. A decrease in ONH blood flow after IOP elevation to 50 and 70 mm Hg was observed in the diabetic animals, whereas the blood flow was well maintained in the healthy animals. Two-way interaction is significant (P = 0.0002, repeated-measures ANOVA), and the differences between the two groups at each assessment time point are significant (*P < 0.05, **P < 0.01, t-tests).
Changes in the mean systemic BP of both groups during the experiment are shown in Table 1A. No significant (P > 0.05, ANOVA) alteration in the BP was observed during the experiment in both groups. The BP of the diabetic animals was not significantly lower (P > 0.05, t-test) than that of the healthy animals at any assessment time point (Table 1A). 
Table 1.
 
Change in Mean Blood Pressure during the Experiment
Table 1.
 
Change in Mean Blood Pressure during the Experiment
A. Change in Both Groups
Baseline 10 min 30 min 20 min 40 min 50 min 60 min P (ANOVA)
Control 94.6 ± 6.9 90.7 ± 4.6 90.6 ± 5.7 91.3 ± 5.1 97.8 ± 2.2 95.7 ± 3.9 96.1 ± 6.4 0.20
Diabetic 88.6 ± 1.9 89.5 ± 6.0 92.1 ± 4.9 87.9 ± 5.6 94.6 ± 2.0 94.6 ± 4.5 94.2 ± 6.4 0.07
B. Change in Each Group
Baseline 10 min 30 min 20 min 40 min 50 min 60 min P (ANOVA)
Control 105.9 ± 14.0 101.3 ± 11.2 97.7 ± 7.9 100.2 ± 9.5 98.0 ± 6.9 97.1 ± 7.3 99.8 ± 5.6 0.36
Octanol 1 mM 108.5 ± 11.1 104.9 ± 12.7 105.1 ± 9.3 103.0 ± 9.0 107.6 ± 5.7 101.4 ± 7.1 106.5 ± 1.3 0.44
Octanol 3 mM 104.3 ± 18.1 105.6 ± 6.1 109.0 ± 6.7 108.2 ± 9.5 104.5 ± 7.6 106.3 ± 5.1 110.8 ± 6.4 0.89
Octanol 10 mM 104.1 ± 10.4 101.9 ± 9.1 97.2 ± 9.8 102.2 ± 11.8 102.5 ± 9.8 101.0 ± 10.0 104.3 ± 10.5 0.92
gap27 114.5 ± 17.1 104.5 ± 9.2 103.3 ± 6.8 105.6 ± 14.1 102.8 ± 8.2 104.5 ± 7.9 104.7 ± 6.4 0.75
The relative NB values (ONH blood flow) are plotted against the OPPs in Figure 2. In healthy animals, the relative NB was well maintained, despite the alteration of the OPP, and linear regression analysis showed that the correlation between them was not significant (P = 0.07, Pearson). In contrast, linear regression analysis showed that the relative NB correlated significantly with the OPP in the diabetic animals (r = 0.71; P < 0.0001, Pearson). The positive correlation indicated that the ONH blood flow decreased when the OPP decreased. 
Figure 2.
 
Results of linear regression analyses between ONH blood flow (NB) and OPP. (A) In healthy animals, the correlation between the ONH blood flow and OPP is not significant (P = 0.07, Pearson). (B) In diabetic rabbits, there is a significant positive correlation (r = 0.71; P < 0.0001, Pearson).
Figure 2.
 
Results of linear regression analyses between ONH blood flow (NB) and OPP. (A) In healthy animals, the correlation between the ONH blood flow and OPP is not significant (P = 0.07, Pearson). (B) In diabetic rabbits, there is a significant positive correlation (r = 0.71; P < 0.0001, Pearson).
Effects of Octanol on ONH Blood Flow
The effects of gap junction uncouplers on the relative NB (ONH blood flow) in response to IOP alterations are shown in Figure 3. In the controls, which received an intravitreal injection of balanced saline solution, the ONH blood flow was maintained at 95.8% ± 7.5% and 86.2% ± 12.2% of the baseline when IOP was elevated to 50 and 70 mm Hg, respectively. Injection of octanol caused a dose-dependent reduction in NB. Octanol at 10 mM decreased NB to 78.0% ± 10.1% and 56.0% ± 12.5% of the baseline when IOP was increased to 50 and 70 mm Hg, respectively (Fig. 3). Two-way, repeated-measures ANOVA showed that the difference between the control and 10 mM octanol-treated eyes was significant (P = 0.002). In addition, there were significant differences from the control at each assessment time point after IOP elevation to 50 and 70 mm Hg (P < 0.01, t-tests). Changes in BP during the experimental period in each group are summarized in Table 1B. No significant (P > 0.05, ANOVA) alterations in the mean BP was observed in each group. The BP was not significantly lower (P > 0.05, t-test) in any treatment group than in the control at any assessment time point (Table 1B). 
Figure 3.
 
Effects of octanol and gap27 on the ONH blood flow in response to IOP elevations. In the control rabbits that received intravitreal injection of balanced saline solution, the ONH blood flow was well maintained. The minimum level was 86.2% ± 12.2% of the baseline after the IOP was elevated to 70 mm Hg. After octanol treatment, the blood flow decreased after IOP elevation in a dose-dependent way. Two-way interaction is significant (P = 0.002, repeated-measures ANOVA) between octanol (10 mM) and the control. Treatment with gap27 caused effects similar to those induced by octanol (10 mM). The ONH blood flow is significantly (P < 0.05, ANOVA) decreased from the control (†P < 0.05, *P < 0.01, **P < 0.001, t tests).
Figure 3.
 
Effects of octanol and gap27 on the ONH blood flow in response to IOP elevations. In the control rabbits that received intravitreal injection of balanced saline solution, the ONH blood flow was well maintained. The minimum level was 86.2% ± 12.2% of the baseline after the IOP was elevated to 70 mm Hg. After octanol treatment, the blood flow decreased after IOP elevation in a dose-dependent way. Two-way interaction is significant (P = 0.002, repeated-measures ANOVA) between octanol (10 mM) and the control. Treatment with gap27 caused effects similar to those induced by octanol (10 mM). The ONH blood flow is significantly (P < 0.05, ANOVA) decreased from the control (†P < 0.05, *P < 0.01, **P < 0.001, t tests).
When the IOP was maintained at 20 mm Hg in animals receiving intravitreal octanol (10 mM), the NB values did not decrease significantly (P > 0.05, one-way ANOVA) and the blood flow was maintained at 89.7% ± 11.7% (n = 5) of the baseline. Thus, the reduction in the ONH blood flow was caused by the elevations of the IOP and not by the direct effect of octanol. 
Linear regression analyses between the relative NB values and OPPs in the eyes receiving balanced saline solution (control) or octanol are shown in Figure 4. In animals that received intravitreal octanol (1.0, 3.0, 10.0 mM), a significant positive correlation was found between the relative NB values and the OPPs. In addition, the correlation coefficients increased dose-dependently. In contrast, the correlation in the controls that received intravitreal balanced saline solution was not significant (P = 0.058, Pearson). 
Figure 4.
 
Linear regression analyses between ONH blood flow (NB) and OPP in eyes treated with octanol and gap27. (A) Control; (B) 1.0 mM octanol; (C) 3.0 mM octanol; (D) 10 mM octanol; (E) 10 μM gap27. In the control receiving intravitreal injection of balanced saline solution, the ONH blood flow is not dependent on OPPs (P = 0.058, Pearson). Octanol (1.0, 3.0, and 10 mM) caused a significant, dose-dependent positive correlation between ONH blood flow and OPP (r = 0.44, 0.62, and 0.71, respectively). The connexin-mimetic peptide gap27 caused effects similar to those induced by octanol (10 mM), where the ONH blood flow is significantly dependent on OPP (P = 0.0005; r = 0.75, Pearson).
Figure 4.
 
Linear regression analyses between ONH blood flow (NB) and OPP in eyes treated with octanol and gap27. (A) Control; (B) 1.0 mM octanol; (C) 3.0 mM octanol; (D) 10 mM octanol; (E) 10 μM gap27. In the control receiving intravitreal injection of balanced saline solution, the ONH blood flow is not dependent on OPPs (P = 0.058, Pearson). Octanol (1.0, 3.0, and 10 mM) caused a significant, dose-dependent positive correlation between ONH blood flow and OPP (r = 0.44, 0.62, and 0.71, respectively). The connexin-mimetic peptide gap27 caused effects similar to those induced by octanol (10 mM), where the ONH blood flow is significantly dependent on OPP (P = 0.0005; r = 0.75, Pearson).
Effects of Gap27 on NB
Similar to octanol, 10 μM of gap27 led to a significant reduction in the NB values (to 70.2% ± 15.6% of the baseline when the IOP was raised to 50 mm Hg and to 42.0% ± 6.9% when the IOP was elevated to 70 mm Hg; Fig. 3). Two-way, repeated-measures ANOVA showed that both decreases were significant (P = 0.003), and the decrease at each time was significant (P < 0.05, t-test). 
In the eyes treated with gap27, a significant positive correlation was found between the relative ONH blood flow and OPPs (r = 0.75; P = 0.0005; Pearson; Fig. 4E). The positive correlation indicated that the ONH blood flow decreased when the OPP was decreased. When the IOP was maintained at 20 mm Hg after the injection of gap27, ONH blood flow did not decrease significantly from the baseline (P = 0.97, one-way ANOVA), and the level was maintained at 90.0% ± 6.0% (n = 4) of the baseline. 
Retinal Function after Intravitreal Injection of Octanol and Gap27
To determine whether the reduction of blood flow by octanol and gap27 was due to functional changes in retinal neurons, we recorded ERGs and VEPs before and after the injections of these gap junction uncouplers. Octanol (10 mM) and gap27 (10 μM) did not cause a significant reduction of amplitudes or prolongation of the implicit times of each parameter of the ERGs and VEPs (Table 2). 
Table 2.
 
Effects of Octanol and gap27 on ERGs and VEPs
Table 2.
 
Effects of Octanol and gap27 on ERGs and VEPs
Before After P *
ERG
Octanol
    a-Wave
        Amplitude, μV 72.7 ± 10.4 86.4 ± 32.6 0.39
        Implicit time, ms 4.3 ± 0.2 4.15 ± 0.25 0.60
    b-Wave
        Amplitude, μV 212.6 ± 40.2 216.0 ± 34.4 0.90
        Implicit time, ms 58.7 ± 3.9 60.6 ± 6.0 0.65
gap27
    a-Wave
        Amplitude, μV 118.1 ± 25.2 128.6 ± 19.7 0.28
        Implicit time, ms 4.1 ± 0.1 4.2 ± 0.1 0.18
    b-Wave
        Amplitude, μV 310.2 ± 63.0 319.4 ± 128.9 0.81
        Implicit time, ms 59.3 ± 4.8 59.9 ± 2.2 0.88
VEP
Octanol
    N1 Implicit time, ms 20.3 ± 1.0 19.8 ± 1.0 0.24
gap27
    N1 Implicit time, ms 20.8 ± 0.7 20.8 ± 0.5 0.78
Discussion
Our results showed that the correlation between NB and OPP in diabetic rabbits was significant. The finding indicates that autoregulation was impaired, and a reduction in blood flow occurred during IOP elevations. Because the administration of two different gap junction uncouplers induced similar changes, the impaired autoregulation in the diabetic rabbits was most likely caused in a large part by a disruption of gap junction communication. Because the blood supply to the ONH of rabbits is somewhat similar to that in humans, 24 the loss of autoregulation in these diabetic animals may explain the clinical observations that diabetes is associated with anterior ischemic optic neuropathy (AION) 25,26 and a faster progression of glaucoma in diabetic eyes. 27  
Hyperpolarization of endothelial cells is transmitted to pericytes and vascular smooth muscle cells through gap junctions by electrically or endothelium-derived hyperpolarizing factors. 28,29 This cell-to-cell communication among vascular cells enables endothelial cells, pericytes, and vascular smooth muscle cells to function as a unit. 29,30 The longitudinal conductance through retinal vessels over a certain distance is important in the coordination of blood flow in the vascular network of the retina. 30 Thus, functional couplings through gap junctions may help in modulating the retinal blood flow to meet the metabolic demands. 6,31,32 This possibility also applies to the vascular system in the ONH, because the vascular system supply within the ONH has characteristics similar to that of the retinal vessels. 33  
The decrease in coupling through gap junctions in the retinal microvessels in diabetic rats may be due to the activation of protein kinase C (PKC). 6 Diabetes and high glucose conditions decrease the expression of Cx43 and cause apoptosis of endothelial cells in retinal vessels. 8,34,35 Thus, disruption of gap junction communication may cause both functional and histologic changes of the retinal vasculature in diabetic eyes. 
Astrocytes are believed to regulate blood flow according to the neuronal activities at gliovascular interfaces in the central nervous system [CNS], where astrocytic network with gap junctions couplings surround the vessel walls. 5 Neuronal activities induce the release of neurotransmitters (e.g., glutamate) and alter the ATP levels. Glutamate stimulates metabotropic glutamate receptors (mGluR) and adenosine activates A2B receptors on astrocytes, resulting in an increase of Ca2+ causing vascular dilatation. 36,37 Imaging studies of the brain have shown that Ca2+ waves spread to neighboring astrocytes, adjusting the local blood flow according to neuronal activity. 38  
This neurovascular coupling may also play a role in regulating blood flow in the retina and ONH. 39 In fact, blood flow regulation in the retina and the ONH is dependent on neuronal activity. 39 41 Astrocytes are the predominant glial cells in the ONH, and astrocytes and Müller cells are the two main glial cells in the retina. 42 Although it remains unclear whether neurons, glial cells, and vascular cells that are coupled by gap junctions, share connexin networks, neuron, and glial cells interfere with each other in the retina. 43 In addition, coupling through gap junctions exists from astrocytes on retinal arteries to Müller cells. 44 Müller cell are also coupled to neighboring cells. 45 Thus, these glial cells may be involved in the neurovascular coupling, where gap junction communication also plays a crucial role. 
To determine the IOP-induced changes in the ONH blood flow with preservation of neuronal activities in the retina and optic nerve, it was necessary to use conscious animals under local anesthesia. The use of conscious animals also helped determine the effects of gap junction uncouplers on the ONH blood flow including neurovascular coupling. Importantly however, because octanol (10 mM) and gap27 (10 μM) did not impair ERGs and VEPs, these chemicals did not block the function of retinal neurons or cause acute retinal damage. Thus, the decrease in the ONH blood flow was not due to loss of neuronal activities in the retina. 
Takayama et al. 14 have demonstrated that the ONH blood flow was well maintained when the IOP was increased from 20 to 50 mm Hg, and the blood flow was decreased by 30% when the IOP was elevated from 20 to 60 mm Hg in rabbits under general anesthesia. Because it has been demonstrated that general anesthesia influences cerebral autoregulation, 46 the use of general anesthesia may account for the discrepancy from our results. Besides, their method of increasing the IOP was more abrupt than ours (i.e., from 20 mm Hg directly to 60 mm Hg) and may also account for the difference. 
One limitation of this study was that we did not determine how the gap junctions were disrupted in the ONH of diabetic rabbits or in the eyes receiving the gap junction uncouplers. However, disruptions of gap junctions are known to occur in diabetic retinal vessels. 6,8,34,35 In addition, neurovascular coupling is impaired in diabetic human retinas. 41 We are not certain which gap junctions are affected by octanol or gap27; however, we used a much lower concentrations of gap27 (10 μM) than the concentration (300 μM) that significantly inhibits acetylcholine-induced relaxation of isolated aorta. 22 Thus, gap27 probably uncoupled gap junctions at multiple sites that connect homogenous and heterogeneous types of vascular, neuronal, and glial cells. Further studies will be directed to resolving this question. 
In conclusion, we suggest that disruptions of gap junction communications disturb blood flow regulation in the ONH, which may account in part for the association of diabetes with AION and glaucoma. 
Footnotes
 Supported by grants from the Japanese Ministry of Education (22591972, 22591973) and the Osaka Eye Bank Association Fund.
Footnotes
 Disclosure: M. Shibata, None; H. Oku, None; T. Sugiyama, None; T. Kobayashi, None; M. Tsujimoto, None; T. Okuno, None; T. Ikeda, None
The authors thank Duco Hamasaki, Bascom Palmer Eye Institute, University of Miami School of Medicine, for editing this manuscript. 
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Figure 1.
 
Changes in the ONH blood flow (or NB) relative to the baseline in response to IOP elevation. Data are expressed as the mean ± SD. A decrease in ONH blood flow after IOP elevation to 50 and 70 mm Hg was observed in the diabetic animals, whereas the blood flow was well maintained in the healthy animals. Two-way interaction is significant (P = 0.0002, repeated-measures ANOVA), and the differences between the two groups at each assessment time point are significant (*P < 0.05, **P < 0.01, t-tests).
Figure 1.
 
Changes in the ONH blood flow (or NB) relative to the baseline in response to IOP elevation. Data are expressed as the mean ± SD. A decrease in ONH blood flow after IOP elevation to 50 and 70 mm Hg was observed in the diabetic animals, whereas the blood flow was well maintained in the healthy animals. Two-way interaction is significant (P = 0.0002, repeated-measures ANOVA), and the differences between the two groups at each assessment time point are significant (*P < 0.05, **P < 0.01, t-tests).
Figure 2.
 
Results of linear regression analyses between ONH blood flow (NB) and OPP. (A) In healthy animals, the correlation between the ONH blood flow and OPP is not significant (P = 0.07, Pearson). (B) In diabetic rabbits, there is a significant positive correlation (r = 0.71; P < 0.0001, Pearson).
Figure 2.
 
Results of linear regression analyses between ONH blood flow (NB) and OPP. (A) In healthy animals, the correlation between the ONH blood flow and OPP is not significant (P = 0.07, Pearson). (B) In diabetic rabbits, there is a significant positive correlation (r = 0.71; P < 0.0001, Pearson).
Figure 3.
 
Effects of octanol and gap27 on the ONH blood flow in response to IOP elevations. In the control rabbits that received intravitreal injection of balanced saline solution, the ONH blood flow was well maintained. The minimum level was 86.2% ± 12.2% of the baseline after the IOP was elevated to 70 mm Hg. After octanol treatment, the blood flow decreased after IOP elevation in a dose-dependent way. Two-way interaction is significant (P = 0.002, repeated-measures ANOVA) between octanol (10 mM) and the control. Treatment with gap27 caused effects similar to those induced by octanol (10 mM). The ONH blood flow is significantly (P < 0.05, ANOVA) decreased from the control (†P < 0.05, *P < 0.01, **P < 0.001, t tests).
Figure 3.
 
Effects of octanol and gap27 on the ONH blood flow in response to IOP elevations. In the control rabbits that received intravitreal injection of balanced saline solution, the ONH blood flow was well maintained. The minimum level was 86.2% ± 12.2% of the baseline after the IOP was elevated to 70 mm Hg. After octanol treatment, the blood flow decreased after IOP elevation in a dose-dependent way. Two-way interaction is significant (P = 0.002, repeated-measures ANOVA) between octanol (10 mM) and the control. Treatment with gap27 caused effects similar to those induced by octanol (10 mM). The ONH blood flow is significantly (P < 0.05, ANOVA) decreased from the control (†P < 0.05, *P < 0.01, **P < 0.001, t tests).
Figure 4.
 
Linear regression analyses between ONH blood flow (NB) and OPP in eyes treated with octanol and gap27. (A) Control; (B) 1.0 mM octanol; (C) 3.0 mM octanol; (D) 10 mM octanol; (E) 10 μM gap27. In the control receiving intravitreal injection of balanced saline solution, the ONH blood flow is not dependent on OPPs (P = 0.058, Pearson). Octanol (1.0, 3.0, and 10 mM) caused a significant, dose-dependent positive correlation between ONH blood flow and OPP (r = 0.44, 0.62, and 0.71, respectively). The connexin-mimetic peptide gap27 caused effects similar to those induced by octanol (10 mM), where the ONH blood flow is significantly dependent on OPP (P = 0.0005; r = 0.75, Pearson).
Figure 4.
 
Linear regression analyses between ONH blood flow (NB) and OPP in eyes treated with octanol and gap27. (A) Control; (B) 1.0 mM octanol; (C) 3.0 mM octanol; (D) 10 mM octanol; (E) 10 μM gap27. In the control receiving intravitreal injection of balanced saline solution, the ONH blood flow is not dependent on OPPs (P = 0.058, Pearson). Octanol (1.0, 3.0, and 10 mM) caused a significant, dose-dependent positive correlation between ONH blood flow and OPP (r = 0.44, 0.62, and 0.71, respectively). The connexin-mimetic peptide gap27 caused effects similar to those induced by octanol (10 mM), where the ONH blood flow is significantly dependent on OPP (P = 0.0005; r = 0.75, Pearson).
Table 1.
 
Change in Mean Blood Pressure during the Experiment
Table 1.
 
Change in Mean Blood Pressure during the Experiment
A. Change in Both Groups
Baseline 10 min 30 min 20 min 40 min 50 min 60 min P (ANOVA)
Control 94.6 ± 6.9 90.7 ± 4.6 90.6 ± 5.7 91.3 ± 5.1 97.8 ± 2.2 95.7 ± 3.9 96.1 ± 6.4 0.20
Diabetic 88.6 ± 1.9 89.5 ± 6.0 92.1 ± 4.9 87.9 ± 5.6 94.6 ± 2.0 94.6 ± 4.5 94.2 ± 6.4 0.07
B. Change in Each Group
Baseline 10 min 30 min 20 min 40 min 50 min 60 min P (ANOVA)
Control 105.9 ± 14.0 101.3 ± 11.2 97.7 ± 7.9 100.2 ± 9.5 98.0 ± 6.9 97.1 ± 7.3 99.8 ± 5.6 0.36
Octanol 1 mM 108.5 ± 11.1 104.9 ± 12.7 105.1 ± 9.3 103.0 ± 9.0 107.6 ± 5.7 101.4 ± 7.1 106.5 ± 1.3 0.44
Octanol 3 mM 104.3 ± 18.1 105.6 ± 6.1 109.0 ± 6.7 108.2 ± 9.5 104.5 ± 7.6 106.3 ± 5.1 110.8 ± 6.4 0.89
Octanol 10 mM 104.1 ± 10.4 101.9 ± 9.1 97.2 ± 9.8 102.2 ± 11.8 102.5 ± 9.8 101.0 ± 10.0 104.3 ± 10.5 0.92
gap27 114.5 ± 17.1 104.5 ± 9.2 103.3 ± 6.8 105.6 ± 14.1 102.8 ± 8.2 104.5 ± 7.9 104.7 ± 6.4 0.75
Table 2.
 
Effects of Octanol and gap27 on ERGs and VEPs
Table 2.
 
Effects of Octanol and gap27 on ERGs and VEPs
Before After P *
ERG
Octanol
    a-Wave
        Amplitude, μV 72.7 ± 10.4 86.4 ± 32.6 0.39
        Implicit time, ms 4.3 ± 0.2 4.15 ± 0.25 0.60
    b-Wave
        Amplitude, μV 212.6 ± 40.2 216.0 ± 34.4 0.90
        Implicit time, ms 58.7 ± 3.9 60.6 ± 6.0 0.65
gap27
    a-Wave
        Amplitude, μV 118.1 ± 25.2 128.6 ± 19.7 0.28
        Implicit time, ms 4.1 ± 0.1 4.2 ± 0.1 0.18
    b-Wave
        Amplitude, μV 310.2 ± 63.0 319.4 ± 128.9 0.81
        Implicit time, ms 59.3 ± 4.8 59.9 ± 2.2 0.88
VEP
Octanol
    N1 Implicit time, ms 20.3 ± 1.0 19.8 ± 1.0 0.24
gap27
    N1 Implicit time, ms 20.8 ± 0.7 20.8 ± 0.5 0.78
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