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Indomethacin lowers optic nerve oxygen tension and reduces the effect of carbonic anhydrase inhibition and carbon dioxide breathing
  1. D B Pedersen1,2,
  2. T Eysteinsson3,
  3. E Stefánsson3,
  4. J F Kiilgaard1,2,
  5. M la Cour1,
  6. K Bang4,
  7. P K Jensen1
  1. 1Department of Ophthalmology, Rigshospitalet, Copenhagen, Denmark
  2. 2University of Copenhagen, Denmark
  3. 3Department of Ophthalmology, University of Iceland, Reykjavik, Iceland
  4. 4Merck, Sharp & Dohme, Glostrup, Denmark
  1. Correspondence to: D B Pedersen MD Department of Ophthalmology, 2061, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen Ø, Denmark; danielladadlnet.dk

Abstract

Background/aims: Prostaglandins are important in blood flow regulation. Carbon dioxide (CO2) breathing and carbonic anhydrase inhibition increase the oxygen tension in the retina and optic nerve. To study the mechanism of this effect and the role of cyclo-oxygenase in the regulation of optic nerve oxygen tension (ONPO2), the authors investigated how indomethacin affects ONPO2 and the ONPO2 increases caused by CO2 breathing and carbonic anhydrase inhibition in the pig.

Methods: Optic nerve oxygen tension was measured in 11 pigs with a polarographic oxygen electrode. The tip of the electrode was placed 0.5 mm above the optic disc. The effects of indomethacin, CO2 breathing (3%) before and after indomethacin treatment, and carbonic anhydrase inhibition with or without indomethacin treatment were investigated.

Results: Administration of 300 mg indomethacin decreased optic nerve oxygen tension significantly. Carbonic anhydrase inhibition and CO2 breathing increased ONPO2 significantly. After indomethacin had been given, the rise in ONPO2 caused by CO2 breathing and carbonic anhydrase inhibition was significantly reduced.

Conclusion: Systemic administration of indomethacin decreases the optic nerve oxygen tension; this is probably the result of decreased blood flow through vasoconstriction of vessels in the optic nerve. Additionally, indomethacin diminishes the ONPO2 increasing effect of CO2 breathing and carbonic anhydrase inhibition, thus affecting the reactivity of vessels in the optic nerve.

  • aPCO2, arterial PCO2
  • apH, arterial pH
  • aPO2, arterial PO2
  • CAIs, carbonic anhydrase inhibitors
  • HR, heart rate
  • MAP, mean arterial pressure
  • NO, nitric oxide
  • ONPO2, optic nerve oxygen tension
  • indomethacin
  • optic nerve
  • oxygen tension
  • carbonic anhydrase inhibition
  • carbon dioxide breathing
  • aPCO2, arterial PCO2
  • apH, arterial pH
  • aPO2, arterial PO2
  • CAIs, carbonic anhydrase inhibitors
  • HR, heart rate
  • MAP, mean arterial pressure
  • NO, nitric oxide
  • ONPO2, optic nerve oxygen tension
  • indomethacin
  • optic nerve
  • oxygen tension
  • carbonic anhydrase inhibition
  • carbon dioxide breathing

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Prostaglandins are important in regulating blood flow in skeletal muscle, stomach, kidney, brain, and retina.1 In the retina of miniature pigs, prostaglandin E1 induces vasodilatation while indomethacin, blocking prostaglandin synthesis, causes vasoconstriction and diminishes the vasodilatory effect of hypoxia and hypercapnia.2 We investigated the role of prostaglandins in regulating optic nerve oxygen tension (ONPO2) in steady state, during hypercapnia, and during carbonic anhydrase inhibition.

Systemic carbonic anhydrase inhibition induces vasodilatation and increases blood flow in the central nervous system3,4 including the retina5 and choroid.6 Carbonic anhydrase inhibitors (CAIs) elevate ONPO2 in pigs7 but the mechanism of action is not known. Systemic administration of CAI causes carbon dioxide (CO2) accumulation in blood and tissue,8 known to increase cerebral blood flow.9 Additionally CO2 breathing causes a similar increase in ONPO2 and arterial PCO2 as do CAIs.7 Therefore, it is likely that CAI works through CO2 to increase ONPO2.

The nitric oxide (NO) pathways probably have an important role in hypercapnic vasodilatation,10 but it is debatable whether prostaglandins are involved in this process.2,11–14 We investigated if blocking the synthesis of prostaglandins affects baseline ONPO2 and the ONPO2 increases caused by CO2 and carbonic anhydrase inhibition.

MATERIALS AND METHODS

Materials

Eleven pigs (Danish Landrace), weighing 28–30 kg were used. Their treatment was supervised by a veterinarian nurse and followed the ARVO resolution for the use of animals in ophthalmic and vision research. Permission for using the pigs was granted by the Danish board for surveillance of the use of experimental animals (Danish Animal Experiments Inspectorate).

Dorzolamide HCl was obtained from Merck, Sharp & Dohme, Glostrup, Denmark; 500 mg was dissolved as a 3% solution in 100 mM citrate buffer, pH 5.6. Indomethacin (Bie & Berntsen, Copenhagen, Denmark) was dissolved in isotonic saline (6 mg/ml) by stirring the solution for 1 hour. Breathing of 3% CO2 was performed by adding CO2 (AGA, Copenhagen, Denmark) to the air, making inspiratory air contain 3% CO2, decreasing nitrogen correspondingly

Anaesthesia and animal preparation

Anaesthesia and animal preparation was performed as previously described.15

Oxygen tension measurements

The optic nerve oxygen tension was measured with a polarographic oxygen electrode with an internal Ag/AgCl reference electrode. The electrode was placed over the optic nerve in the pig as previously described.15

Experimental protocol

Arterial blood samples were drawn from the catheter in the femoral artery and analysed for arterial oxygen tension (aPO2), arterial carbon dioxide tension (aPCO2), and arterial pH (apH) using an ABL 605 blood gas analyser (Radiometer, Copenhagen, Denmark).

Normal values for aPO2 (10–14 kPa), aPCO2 (5.5–7.5 kPa), and apH (7.38–7.42) were ensured by adjusting the respirator.

Initially a stable oxygen recording in the vitreous was obtained. Then the pig was given 100% oxygen in the inspiratory air ensuring that the oxygen electrode worked satisfactorily. Following this the pig was ventilated with atmospheric air, except when CO2 breathing was performed.

Figure 1 shows the course of the experiment for the pigs given indomethacin (n = 5). The control pigs (n = 6) were given 500 mg dorzolamide.

Figure 1

 The course of the experiment. Initially 3% CO2 breathing is induced (control; n = 4); 30 minutes later, the breathing air is changed back to room air. After having reached baseline, 300 mg indomethacin is injected intravenously (n = 5). One hour later, 3% CO2 breathing is started again (indomethacin treated pigs; n = 4). After 30 minutes of room air, 500 mg dorzolamide is injected (n = 5).

The mean total duration of the experiments was 6 hours and 15 (SD 66) minutes (n = 11). The oxygen electrode drift was less than −0.1 kPa/h.

Data processing

ONPO2, mean arterial pressure (MAP) and heart rate (HR) values (a window of 15 seconds of the recording was averaged with the Axoscope program) for baseline and time points after injection of the study drugs were used for all the calculations.

The mean and standard deviation of ONPO2, apH, aPCO2, aPO2, MAP, and HR were calculated and presented in table 1.

Table 1

 Optic nerve oxygen tension (ONPO2), arterial pH (apH), arterial PCO2 (aPCO2), arterial PO2 (aPO2), mean arterial pressure (MAP), and heart rate (HR) before and at a time point after CO2 breathing, indomethacin injection, or dorzolamide injection. The chosen time points are 60 minutes after the injection of indomethacin and 30 minutes after beginning of CO2 breathing and carbonic anhydrase inhibition

Differences in the parameters before and after drug injections in each pig were tested for significance using Student’s paired two tailed t test (table 1). Differences in the ONPO2 changes between groups were tested for significance using Student’s unpaired two tailed t test. A significance level of 0.05 was used.

Graphic presentations of the tracings were made in SigmaPlot 2001 for Windows (Version 6.00 SPSS Inc, Chicago, IL, USA).

RESULTS

The effect of indomethacin on ONPO2

Before injection of indomethacin, baseline optic nerve oxygen tension was 2.96 (0.59) kPa (n = 5). This was significantly decreased by −1.22 (0.43) kPa (p = 0.002) 1 hour after administration of 300 mg indomethacin (table 1 and fig 2A). All other parameters had not changed significantly 60 minutes after the injection (table 1).

Figure 2

 Recording of the delta optic nerve oxygen tension in kPa over time during different kinds of manipulations. Baseline ΔONPO2 (before the beginning of the manipulation) is calibrated to 0 kPa. (A) The effect of injection of 300 mg indomethacin at time 0. (B) The effect of 3% CO2 breathing at time 0 before and after indomethacin treatment. (C) The effect of injection of 500 mg dorzolamide at time 0 in indomethacin treated animals and in controls.

The effect of CO2 breathing on ONPO2 before and after indomethacin treatment

ONPO2 was significantly increased by 0.66 (0.23) kPa (n = 4, p = 0.002) 30 minutes after the start of 3% CO2 breathing (table 1, figs 2B and 3). After indomethacin administration, 3% CO2 breathing caused an insignificant increase in ONPO2 (p = 0.27) (table 1, figs 2B and 3). This increase was significantly smaller than before administration of the drug (p = 0.029) (fig 3). One experiment with CO2 breathing was excluded because of movement of the pig head; however, the measurements during the following injection of dorzolamide could be performed satisfactorily and was included.

Figure 3

 The increase in optic nerve oxygen tension 30 minutes after beginning of 3% CO2 breathing (mean (SD)) in control pigs (n = 4) and indomethacin treated pigs (n = 4). *Indicates a significant increase (Student’s paired t test, p<0.05). The Δ values are compared by using a Student’s unpaired t test.

The effect of CAI on ONPO2 with and without indomethacin treatment

Dorzolamide 500 mg increased ONPO2 significantly by 0.73 (0.34) kPa (n = 6, p = 0.004) in the untreated pigs (table 1, figs 2C and 4). In the indomethacin treated pigs an intravenous injection of 500 mg dorzolamide increased ONPO2 by 0.22 (0.12) kPa (n = 5, p = 0.015) (table 1, figs 2C and 4). This was a significantly smaller increase than in the controls (p = 0.013) (fig 4), despite similar changes in apH and aPCO2 in the animals (table 1).

Figure 4

 The increase in optic nerve oxygen tension 30 minutes after 500 mg dorzolamide iv (mean (SD)) in control pigs (n = 6) and indomethacin treated pigs (n = 5). *Indicates a significant increase (Student’s paired t test, p<0.05). The Δ values are compared by using a Student’s unpaired t test.

DISCUSSION

In this study two new findings were done. Firstly, indomethacin injected intravenously decreased optic nerve oxygen tension. Secondly, the ONPO2 increasing effect of CO2 breathing and of carbonic anhydrase inhibition was reduced in indomethacin treated animals when compared with controls.

The indomethacin induced decrease in ONPO2 is probably caused by vasoconstriction and thereby decreased blood flow in the optic nerve. Indomethacin inhibits the production of prostaglandins.16 The fact that indomethacin decreases ONPO2 suggests that prostaglandins have a constant vasodilatory tonus on retinal vessels, as has NO.17 Myography studies have shown that derivatives of arachidonic acids such as prostaglandin F, prostaglandin E2, nd thromboxane2 have a vasoconstrictory tonus on calf retinal arterioles, whereas prostacyclin (prostaglandin I2) has a vasodilatory tonus.18 The same types of study found a weak contraction in human ciliary arteries induced by indomethacin.19 Our study indicates that the vasodilatory tonus of the prostaglandins on vessels in the optic nerve is more important than their vasoconstrictory tonus.

Perfusion with indomethacin of the eye of minipigs constricts retinal arterioles,20 but despite continuous perfusion of indomethacin, this effect disappears after 10 minutes. In our experiments, however, the effect lasted the whole period of observation (maximum 70 minutes) (fig 2A). The blood flow decreasing effect of indomethacin lasts for at least 150 minutes in the human brain.21 Systemic administration of indomethacin also decreases cerebral blood flow in rabbits,1 rats,22 newborn pigs,23,24 and humans.21,25 However, Cuypers et al did not find this effect in the rabbits they examined.26

We found that indomethacin decreased the ONPO2 increasing effect of CO2. We interpret this to be caused by a reduced CO2 reactivity of optic nerve blood vessels. Similar findings have been reported for the cerebral vessels in rabbits14 and humans.21,25 However, others have not found any effect of indomethacin on cerebral vessel CO2 reactivity in cats12 and rabbits.26

In our experiments, indomethacin also decreased the effect of CAI on ONPO2. A similar effect of indomethacin on CAI reactivity has been reported in rat cerebral vessels.11 Since indomethacin reduced the ONPO2 increase induced by both CO2 breathing and carbonic anhydrase inhibition and since both of these manipulations cause CO2, accumulation it is likely that the effect of carbonic anhydrase inhibition on ONPO2 is caused by the effect of CO2 accumulation.

There are no qualitative differences between the effects of the different cyclo-oxygenase inhibitors27; therefore, other types of NSAID may also decrease the oxygen tension in the optic nerve. However, it has been shown that aspirin does not decrease cerebral blood flow in humans.21,25 Also diclofenac does not decrease the cerebral blood flow in rats while ibuprofen does.11 Ibuprofen decreases pial vessel diameter in newborn pigs but does not affect the CO2 reactivity of these vessels.24 Indomethacin may therefore change the vessel reactivity through affecting other processes than the prostaglandin synthesis,27 but it may also be a more potent drug in inhibiting cyclo-oxygenase.28 The inhibiting effect of indomethacin on CO2 reactivity in cerebral vessels have been reversed by directly adding prostaglandins.24 Thus, it is likely that prostaglandins at least facilitate CO2 induced vasodilation.24

We have previously shown that measurements of the oxygen tension in the normal pig vitreous can be obtained using the 20 μm oxygen electrodes, employed in the present study.7,15 The vitreous acts as a diffusion medium with very low metabolism and oxygen gradient (about 2 mm Hg per mm in vitreous). Thus, the measurements in front of the optic nerve and retina are relatively insensitive to the exact location of the sensor. Also, the oxygen molecule diffuses freely from the retinal vessels to the retina and to the vitreous. Therefore, we interpret the oxygen measurements in the vitreous as reflecting the average oxygen concentration in the vitreal part of the retina and optic nerve head denoted optic nerve oxygen tension and retinal oxygen tension.

Intravenously injected indomethacin decreases ONPO2 in pigs. Also indomethacin diminishes the increase in ONPO2 induced by carbonic anhydrase inhibition and CO2 breathing, indicating that the regulation of the vessels is impaired. This may be relevant with regard to patients with ischaemic diseases in the retina and optic nerve treated with indomethacin or other NSAIDs, since not only may a vasoconstriction be happening but also the ischaemic areas of the tissues may lose the important vasodilatory effect of CO2. The effects of different kinds of NSAIDs have been investigated, and the results have been contradictory.11,21,24,25 Further investigations are needed to clarify whether other NSAIDs have the same effect as indomethacin and also if the drugs orally or topically administered affect optic nerve oxygen tension.

Acknowledgments

The authors thank veterinary technician Mette Olesen, veterinary nurse Letty Klarskov and other staff at the Experimental Surgry Unit, Panum Institute, University of Copenhagen for their assistance with the handling of the experimental animals.

REFERENCES

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Footnotes

  • Supported by University of Copenhagen, Værn om Synet, Denmark, MSD Inc, Denmark, University of Iceland Research Fund, and Helga Jónsdóttir and Sigurlioi Kristjánsson memorial Fund, Iceland.

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