This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsujikawa, A. Articles by Dawson, V. L. Search for Related Content PubMed PubMed Citation Articles by Tsujikawa, A. Articles by Dawson, V. L.

(Stroke. 1998;29:1431-1438.)
© 1998 American Heart Association, Inc.

Original Contributions

Tacrolimus (FK506) Attenuates Leukocyte Accumulation After Transient Retinal Ischemia

Akitaka Tsujikawa, MD; Yuichiro Ogura, MD; Naoko Hiroshiba, MD; Kazuaki Miyamoto, MD; Junichi Kiryu, MD; Yoshihito Honda, MD

From the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.

Correspondence to Akitaka Tsujikawa, MD, Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8507, Japan. E-mail tujikawa{at}capricorn.kuhp.kyoto-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Background and Purpose—Tacrolimus, an immunosuppressant agent, has been shown to reduce tissue injury and leukocyte accumulation after transient ischemia. This study was designed to evaluate quantitatively the inhibitory effects of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation in vivo after transient retinal ischemia and the protective effects of tacrolimus on ischemia-induced neural damage.

Methods—Retinal ischemia was induced for 60 minutes in anesthetized pigmented rats by temporary ligation of the optic sheath. Tacrolimus was administered at 10 minutes after ischemic induction. At 4, 12, 24, and 48 hours after reperfusion, leukocyte behavior in the retinal microcirculation was evaluated in vivo with acridine orange digital fluorography. After 7 days of reperfusion, ischemia-induced retinal damage was evaluated histologically.

Results—Treatment with tacrolimus suppressed leukocyte rolling; the maximum number of rolling leukocytes was reduced by 60.1% at 12 hours after reperfusion (P<0.05). In tacrolimus-treated rats, the velocity of rolling leukocytes was significantly faster than in vehicle-treated rats (P<0.01). The subsequent leukocyte accumulation was reduced by 61.6% at 24 hours after reperfusion (P<0.01). Histological examination demonstrated the protective effect of tacrolimus on ischemia-induced retinal damage, which was more substantial in the inner retina (P<0.01).

Conclusions—The present study demonstrated the inhibitory effect of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation and the therapeutic potency to neural injury after transient retinal ischemia.

Key Words: ischemia • leukocytes • retina • rheology

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Arecent in vivo study demonstrated that tacrolimus ameliorated skilled motor deficits produced by middle cerebral artery occlusion.¹ Because tacrolimus was reported to have a neuroprotective effect,² it has attracted a great deal of attention as a potent agent for treatment of stroke.³ Its potency is supported by in vitro evidence that tacrolimus showed a remarkable protective effect against excitotoxic neural death in cultured cells.^{4 5} Moreover, tacrolimus has been indicated to have such an intense immunosuppressant property that it has been used for the prevention of allograft rejection.⁶ The immunosuppressant property of tacrolimus is initially exerted on leukocytes, especially on T cells.⁷ Many in vivo investigations have indicated that tacrolimus ameliorates allograft rejection by inhibiting leukocyte participation and activation.⁸ When considering tacrolimus as treatment for stroke, its immunosuppressant property to leukocytes would substantially contribute to a neuroprotective effect on stroke. However, little is known about the effect of tacrolimus on leukocyte dynamics in postischemic tissue, especially in the brain.

Because leukocytes are thought to play a central role in ischemia reperfusion injury,⁹ it would be very valuable to investigate leukocyte dynamics in the brain after stroke.¹⁰ But only a few techniques allow an investigation of leukocyte dynamics in the postischemic brain: observation from a cranial window with an intravital microscope¹¹ or a confocal laser scanning microscope.^{12 13} Intravital microscopic observation from a cranial window can visualize leukocyte dynamics only in the pial microcirculation.¹¹ A confocal laser scanning microscope allows us to evaluate leukocyte movement in the cerebrum,¹² but it can make the optical sectioning of the outer 150 µm of the cortex.¹³ Accordingly, these methods cannot visualize leukocyte dynamics deep in the cortex or in the major cerebral vessels.

In contrast to the cerebrum, the optic media (which consists of cornea, lens, vitreous, and retina) are so transparent that the retinal microcirculation could be observed noninvasively in vivo. Moreover, the retina belongs to the central nervous system, and the property seen in endothelial cells and neural cells in the retina is reportedly similar to that in the cerebrum.^{14 15 16} Therefore, investigation of leukocyte dynamics in the postischemic retina would be valuable for the evaluation of leukocyte involvement in the postischemic cerebral injury.

Recently, we have developed the technique of acridine orange digital fluorography.^{17 18 19 20} Acridine orange digital fluorography allows us to visualize leukocytes clearly and to evaluate leukocyte dynamics quantitatively in the retinal microcirculation in vivo. Using this technique, we recently have shown that leukocyte dynamics in the postischemic retina, such as leukocyte rolling, adhesion, and accumulation, could be evaluated quantitatively.²¹ The purpose of this study was to evaluate quantitatively the inhibitory effects of tacrolimus on leukocyte rolling and on subsequent leukocyte accumulation in vivo in the postischemic retina and the therapeutic efficacy of tacrolimus on ischemia-induced retinal damage.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Animal Model
Induction of transient retinal ischemia was reported previously, in which the method described by Stefansson et al,²² with slight modification, was used.^{21 23} Male pigmented Long-Evans rats (200 to 250 g; n=72) were anesthetized with xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (10 mg/kg). The pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. After a lateral conjunctival peritomy and disinsertion of the lateral rectus muscle, the optic sheath of the right eye was exposed by blunt dissection. A 6–0 nylon suture was passed around the optic sheath and tightened until blood flow ceased in all retinal vessels. The absence of perfusion for a 60-minute period was confirmed through an operating microscope, and the suture was removed thereafter. Reperfusion of the vessels was observed through the operating microscope.

Tacrolimus-treated rats were injected intramuscularly with 3.2 mg/kg of tacrolimus at 10 minutes after induction of ischemia; vehicle-treated rats were given the same volume of saline. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Acridine Orange Digital Fluorography
Acridine orange digital fluorography has been previously described elsewhere.^{17 18 19} In this technique, a scanning laser ophthalmoscope (Rodenstock Instrument), coupled with a computer-assisted image analysis system, makes continuous high-resolution images of fundus stained by the metachromatic fluorochrome AO (Wako Pure Chemical). The dye emits a green fluorescence when it interacts with DNAs. The spectral properties of AO-DNA complexes are very similar to those of sodium fluorescein, with an excitation maximum at 502 nm and an emission maximum at 522 nm.²⁴ The argon blue laser was used for the illumination source, with a regular emission filter for fluorescein angiography. Immediately after AO solution was infused intravenously, leukocytes were stained selectively among circulating blood cells. Nuclei of vascular endothelial cells also were stained. The obtained images were recorded on an S-VHS videotape at the video rate of 30 frames/s for further analysis.

Experimental Design
Acridine orange digital fluorography was performed at 4, 12, 24, and 48 hours after reperfusion both in tacrolimus-treated and vehicle-treated groups. Nonischemic rats were evaluated as the control. Six different rats were used at each time point in each group.

Immediately before acridine orange digital fluorography, rats were anesthetized with the same agent, and the pupils were dilated. A contact lens was used to retain corneal clarity throughout the experiment. Each rat had a catheter inserted into the tail vein, and was placed on a stereotaxic platform. Body temperature was maintained at 38±0.5°C throughout the experiment. Arterial blood pressure was monitored with a blood pressure analyzer (IITC) (see the Table). AO (0.1% solution in saline) was injected continuously through the catheter for 1 minute at a rate of 1 mL/min. The fundus was observed with the scanning laser ophthalmoscope in the 40° field for 5 minutes. At 30 minutes after the injection of AO, the fundus was observed again to evaluate leukocytes accumulated in the retinal microcirculation.

View this table: [in this window] [in a new window]   Table 1. Mean Arterial Blood Pressure and Periphereal Leukocyte Count for All Groups

After the experiment, the rat was killed with an anesthetic overdose, and the eye was enucleated to determine a calibration factor with which to convert values measured on a computer monitor (in pixels) into real values (in micrometers).

Image Analysis
The video recordings were analyzed with an image analysis system, which has been described in detail elsewhere.^{17 18 19} In brief, the system consists of a computer equipped with a video digitizer (Radius). The latter digitizes the video image in real time (30 frames/s) to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps. We evaluated the diameters of major retinal vessels, the flux of rolling leukocytes along the major retinal veins, the velocity of rolling leukocytes, and the number of leukocytes accumulated in the retinal microcirculation through use of this system.^{20 21}

The diameters of major retinal vessels were measured at 1 disk diameter from the center of the optic disk in monochromatic images recorded before AO injection. Each vessel diameter was calculated in pixels as the distance between the half-height points determined separately on each side of the density profile of the vessel image and converted into real values using the calibration factor. The averages of the individual arterial and venous diameters were used as the arterial and venous diameters for each rat.

Rolling leukocytes were defined as leukocytes that moved at a velocity slower than that of free-flowing leukocytes. The number of rolling leukocytes was calculated from the number of cells crossing a fixed area of the vessel at a distance 1 disk diameter from the optic disk center per minute. The flux of rolling leukocytes was defined as the total number of rolling leukocytes along all major veins.

Velocity of rolling leukocytes was calculated as the time required for a leukocyte to travel a given distance along the vessel. The average of at least 10 velocities was defined as the velocity of rolling leukocytes for each rat.

The number of leukocytes accumulated in the retinal microcirculation was evaluated at 30 minutes after AO injection. The number of fluorescent dots in the retina within 8 to 10 areas of 100 pixels square at a distance of 1 disk diameter from the edge of the optic disk was counted. The average number of individual areas was used as the number of leukocytes accumulated in the retinal microcirculation for each rat.

Histological Procedures
Six eyes from 6 rats in tacrolimus-treated, vehicle-treated, and nonoperated control groups were obtained to evaluate the severity of retinal damage. After 7 days of reperfusion, the rats were killed with an anesthetic overdose. The operated eyes were immediately enucleated and fixed in 1.48% formaldehyde and 1% glutaraldehyde in phosphate buffer and in 3.7% formaldehyde afterward. Then the eyes were dehydrated, embedded in paraffin, sectioned with a microtome at 4 µm thickness, and stained with hematoxylin and eosin. Each section was cut along the horizontal meridian of the eye through the optic nervehead. Sections were cut perpendicular to the retinal surface. Retinal sections were examined with an optical microscope (x400) to a masking procedure and then digitized by a charge-coupled device camera on a computer monitor.

To quantify the retinal damage induced by ischemia-reperfusion injury, we measured changes in thickness and cell densities of the retina, using the method described by Hughes²⁵ with a slight modification.^{26 27} The thickness of the IPL, INL, ONL, and the overall retina from outer to inner limiting membrane (OLM-ILM) was measured. The thickness of retinal layers in each section was measured in the retina at a distance of 1.5 mm from the center of the optic nerve head. The value of each retinal thickness was averaged from 10 measurements of 4 sections from each eye. In addition, the number of cell nuclei of 3 retinal layers (GCL, INL, and ONL) was counted in the retina of a 50-µm-wide band from both hemispheric sections at a distance of 1.5 mm from the center of the optic nerve head. The value of density of each layer was averaged from measurements of both hemispheres of 4 sections.

Statistical Analysis
All values are mean±SEM. Student's t test was used to compare 2 groups. ANOVA was used to compare 3 or more conditions, with post hoc comparisons tested using the Fisher protected least significant difference procedure. Differences were considered statistically significant when the probability values were less than 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Diameters of Major Retinal Vessels
Figure 1 shows characteristic fundus images of a nonoperated control rat and vehicle-treated rats at various time points after reperfusion. Figure 2 indicates changes of major retinal vessel diameters in control animals and operated rats at various time points after reperfusion. In arteries, significant vasoconstriction occurred immediately after reperfusion (67.7% to 80.5%, P<0.05 versus control rats), with subsequent minor vasodilation. Moreover, vasoconstriction in arteries was more remarkable in tacrolimus-treated rats throughout the experiment compared with vehicle-treated rats (P=0.039). Postischemic veins showed significant vasoconstriction at 4 hours after reperfusion (84.0% in tacrolimus-treated rats and 86.2% in vehicle-treated rats, P<0.05 versus control rats). In contrast, significant vasodilation occurred and peaked at 24 hours after reperfusion (127% in tacrolimus-treated rats and 131% in vehicle-treated rats, P<0.05 versus the values at 4 hours after reperfusion) and subsided at 48 hours after reperfusion. Venous vasodilation in tacrolimus-treated rats was significantly suppressed at all time points after reperfusion compared with that in vehicle-treated rats (P=0.032).

View larger version (141K): [in this window] [in a new window]   Figure 1. Digitized monochromatic images of major retinal vessels obtained with a scanning laser ophthalmoscope in a nonoperated control rat (A) and vehicle-treated rats at 4 (B), 24 (C), and 48 (D) hours after reperfusion. Arteries and veins both showed significant vasoconstriction immediately after reperfusion, which peaked at 4 hours after reperfusion. Subsequent vasodilation occurred and peaked at 24 hours after reperfusion and subsided at 48 hours after reperfusion.

View larger version (18K): [in this window] [in a new window]   Figure 2. Time course of major retinal arterial (A) and venous (B) diameters after reperfusion in tacrolimus-treated and vehicle-treated rats. Values are mean±SEM *P<0.01, P<0.05 compared with values of control rats, P<0.01, §P<0.05 compared with values at 4 hours after reperfusion.

Rolling Leukocytes
Immediately after AO was infused intravenously, leukocytes were stained selectively among circulating blood cells (Figure 3A). Among many free-flowing leukocytes, in operated rats some leukocytes were observed slowly rolling along major retinal veins but not along any major retinal arteries throughout the experiments (Figure 3B and 3C).

View larger version (129K): [in this window] [in a new window]   Figure 3. A, Leukocytes were stained selectively among circulation blood cells. Nuclei of vascular endothelial cells also stained. Leukocytes in the capillaries (arrows) moved slowly, making brief stops; leukocytes in the veins (arrowheads) moved at a higher velocity. B, Among free-flowing leukocytes, many rolling leukocytes were observed along the major retinal veins (arrowheads). No rolling leukocytes were seen along the major retinal arteries. Some leukocytes adhered to the venous wall (arrows). C, Two leukocytes were observed rolling along the venous wall (arrowheads), and 1 leukocyte was adhered to the venous wall (arrow).

In vehicle-treated rats, a small number of leukocytes was observed rolling along the venous walls at 4 hours after reperfusion. The flux of rolling leukocytes substantially increased and peaked at 12 hours after reperfusion. In tacrolimus-treated rats, leukocyte rolling was significantly inhibited (P=0.001) (Figure 4A). The numbers of rolling leukocytes in tacrolimus-treated rats were significantly reduced—by 68.4% (P=0.003), 60.1% (P=0.013), and 48.5% (P=0.026) at 4, 12, and 24 hours after reperfusion, respectively, compared with those in vehicle-treated rats. Figure 4B indicates changes of leukocyte rolling velocity at various time points after reperfusion. The velocity of rolling leukocytes at 12 hours after reperfusion was significantly slower (P=0.001) compared with values from other times. In tacrolimus-treated rats, the velocity of rolling leukocytes was significantly faster than in vehicle-treated rats (P=0.001).

View larger version (17K): [in this window] [in a new window]   Figure 4. A, Time course of flux of rolling leukocytes after reperfusion in tacrolimus-treated and vehicle-treated rats. B, Velocities of rolling leukocytes at each time point after reperfusion in tacrolimus-treated and vehicle-treated rats. Values are mean±SEM. *P<0.01, P<0.05 compared with vehicle-treated rats; P<0.01 compared with other time points.

Leukocytes Accumulated in the Retinal Microcirculation
AO easily infiltrates through the vessel wall and diffuses into the retina because of the permeability of the membrane. Accordingly, a few minutes after AO injection was stopped, fluorescence of circulating leukocytes was faint because of the washout. In contrast, leukocytes that accumulated in the retina remained fluorescent for about 2 hours. These leukocytes were recognized as distinct fluorescent dots at 30 minutes after AO injection, although no circulating leukocytes fluoresced (Figure 5).

View larger version (175K): [in this window] [in a new window]   Figure 5. Leukocytes accumulated in the retina were observed as fluorescent dots at 30 minutes after AO injection. A small number of leukocytes could be found in control rats (A). Increasing numbers of leukocytes accumulated at 4 (B) and 12 (C) hours after reperfusion in vehicle-treated rats and peaked at 24 hours after reperfusion (D). Significant reduction of leukocyte accumulation was seen in the tacrolimus-treated rats at 12 (E) and 24 (F) hours after reperfusion.

Figure 6 indicates changes in numbers of leukocytes accumulated in the retinal microcirculation in both groups. In vehicle-treated rats, few leukocytes could be recognized in the control animals, while accumulated leukocytes began to increase with time after reperfusion and peaked at 24 hours after reperfusion. The number of accumulated leukocytes significantly decreased in tacrolimus-treated rats (P=0.0001). The numbers of accumulated leukocytes were reduced by 35.6% (P=0.019) and 61.6% (P=0.0001) at 12 and 24 hours after reperfusion, respectively, as a result of treatment with tacrolimus.

View larger version (22K): [in this window] [in a new window]   Figure 6. Time course of the number of leukocytes accumulated in the retina after reperfusion in tacrolimus-treated and vehicle-treated rats. Values are mean±SEM. *P<0.01 compared with control rats; P<0.01, P<0.05 compared with vehicle-treated rats.

Retinal Damage
Histological examination showed the destruction of retinal structures and the decrease of retinal thickness in operated rats whether they were treated with tacrolimus or not (Figure 7). The decrease in retinal thickness and damage of retinal cells were more severe in the inner retina, with less obvious changes in the outer retina. Although retinal thickness was reduced both in tacrolimus-treated and vehicle-treated rats, each retinal layer was significantly preserved in tacrolimus-treated rats compared with vehicle-treated rats (P=0.0001). Moreover, cell density of each retinal layer showed that retinal cells were lower in vehicle-treated rats than in tacrolimus-treated rats (P=0.023). The protective effect was more substantial in the inner retina. The thicknesses of INL and IPL in rats treated with tacrolimus were 18.1±2.1 and 16.6±2.1 µm, which was 208% (P=0.0007) and 319% (P=0.0002) of that in vehicle-treated rats (Figure 7). In addition, the cell density of GCL in rats administered tacrolimus was 6.6±0.3 per 50-µm-wide band, which was about 2.6 times as many as that in vehicle-treated rats (P=0.0001).

View larger version (25K): [in this window] [in a new window]   Figure 7. A, Thickness of different retinal layers at 7 days after reperfusion in tacrolimus-treated and vehicle-treated rats. B, Cell density of different retinal layers at 7 days after reperfusion in tacrolimus-treated and vehicle-treated rats. OLM indicates outer limiting membrane; ILM, inner limiting membrane. Values are mean±SEM. *P<0.01, P<0.05 compared with values of control rats; P<0.01, §P<0.05 compared with vehicle-treated rats.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
In this study, we clearly visualized and quantitatively evaluated leukocyte rolling along the retinal vessels and leukocyte accumulation in the retina after transient retinal ischemia in vivo. Increasing amounts of evidence had indicated that leukocytes play a central role in postischemic neural damage in the brain.^{10 28 29} Plugged leukocytes have been suggested to contribute to the no-reflow phenomenon.²⁸ In addition, accumulated leukocytes have been shown to cause tissue injury by producing superoxide radicals³⁰ and various kinds of inflammatory cytokines.³¹ Recently, from experiments investigating leukocyte adhesion to the vascular endothelium, leukocyte recruitment to the inflammatory region has been shown to be mediated through a multistep process.³² Each process is mediated with distinct adhesion molecules and regulated elaborately.³³ Therefore, it would be very valuable to investigate leukocyte dynamics in the cerebrum after transient ischemia.^{10 28 29 34}

Unfortunately, only a few techniques allow us to investigate the cerebral microcirculation.^{11 12 13} With increased spatial resolution and depth penetration, confocal laser scanning microscopy permits the evaluation of leukocyte dynamics in the microcirculation of the pia mater and the outer layers of the cerebral cortex.¹² Surrounded by the cranium, however, a cranial window is a prerequisite to observe the cerebral microcirculation in vivo. In contrast, acridine orange digital fluorography is a noninvasive technique,^{17 18 19 20} except for when retinal ischemia is induced.²¹ In this technique, a scanning laser ophthalmoscope makes continuous, high-resolution images of leukocytes stained by a metachromatic fluorochrome of acridine orange at the rate of 30 frames/s. An image analysis system digitizes the video images to 640 horizontal and 480 vertical pixels with an intensity resolution of 256 steps. While ischemic induction entails a minor surgical preparation, it does not invade the inner structures of the eyeball.^{21 22 23} Preliminary experiments revealed that sham-operated rats, which were subject to the same operation except tightening of the suture, showed no differences in the retinal microcirculation compared with nonoperated control rats.

We investigated the effects of administering tacrolimus on the leukocyte dynamics and neural damage in the postischemic retina. Dirnagl et al¹² have previously reported quantitative evaluation of leukocyte dynamics at the early phase of the postischemic brain. A histological study by Zhang et al¹⁰ demonstrated that neutrophil accumulation in the rat cerebrum after 2 hours of ischemia peaked at 24 to 48 hours after reperfusion. Similarly, we have reported previously that leukocyte rolling and accumulation peaked at 12 and 24 hours after reperfusion, respectively, in the retina subjected to 60 minutes of ischemia.²¹ Therefore, prolonged evaluation should be performed to evaluate the inhibitory effect of agents on leukocyte dynamics after transient ischemia.

Previous studies have indicated that tacrolimus is a potent neuroprotectant in focal and global cerebral ischemia,^{1 3} of which neuroprotective mechanisms remain unclear. Tacrolimus-forming complex with immunophilin inhibits a calcium/calmodulin-dependent phosphatase calcineurin.³⁵ Tacrolimus has been reported to inhibit the calcinulin-induced dephosphorylation and activation of neural NOS and to reduce N-methyl-D-aspartate–mediated neurotoxicity.^{4 5} Furthermore, it has recently been reported that calcineurin-induced dephosphorylation involved in Ca²⁺-triggered apoptosis³⁶ and that tacrolimus may be neuroprotective against delayed neural death by inhibiting Ca²⁺-mediated cell death.³

In addition to these neuroprotective properties, calcineurin inactivation contributes to IL-2 inhibition, interferon gamma expression, and subsequent T-cell proliferation.³⁷ Furthermore, the transcription factor NF-B, which induces several proinflammatory cytokines, is shown to be inhibited by calcineurin inactivation.^{38 39} An increased expression of proinflammatory cytokines such as IL-1, IL-2, TNF-, and interferon gamma has been found after stroke.⁴⁰ Many in vitro studies have shown that stimulation with these inflammatory cytokines upregulates the expression of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1,⁴¹ and selectins.⁴² Treatment with tacrolimus at 10 minutes after induction of ischemic insult significantly inhibited leukocyte rolling along the retinal veins, moreover, it significantly reduced leukocyte accumulation in the postischemic retina. Tacrolimus administration reduced the maximum number of rolling leukocytes at 12 hours after reperfusion by 60.1%. Subsequent leukocyte accumulation in the retina was reduced to 38.4% at 24 hours after reperfusion. In a histological study in the liver, tacrolimus treatment significantly suppressed expression of ICAM-1 at 12 hours after reperfusion.⁸ Immunosuppressant properties would account for a reduction of leukocyte accumulation and the suppression of adhesion molecule expression.

Histological sections demonstrated ischemia-induced tissue damage in operated rats, which was obvious in the inner retina. Histological results in this study showed the protective effect of tacrolimus on neural cell damage after transient ischemia. Indeed, suppression of excitotoxic neural cell death or apoptosis derived from calcineurin inactivation would contribute to this neuroprotective effect.^{4 5} However, in addition to neural cell death induced by ischemic insult accumulated leukocytes would be involved in postischemic tissue injury by blockage of blood flow²⁸ or by the production of reactive oxygen species³⁰ and proinflammatory cytokines that accelerate the inflammation.³¹ Tacrolimus might attenuate retinal damage in part through reductions of leukocyte accumulation and activation.^{8 9} It has been reported that immunosuppression by sublethal whole body X-irradiation resulted in a significant reduction of brain edema and leukopenia after middle cerebral artery occlusion.³⁴ Furthermore, leukopenia induced by an anticancer drug has been reported to reduce infarct volumes after transient cerebral ischemia.²⁹ Although this study did not show that tacrolimus treatment suppressed leukocyte activation, leukocyte accumulation after transient ischemia was significantly ameliorated with tacrolimus treatment. This amelioration would partially contribute to the neuroprotection seen after transient ischemia.

In the present study, as Figure 2 shows, significant vasoconstriction and subsequent vasodilation occurred. Vascular endothelium is a main source of relaxing (eg, NO) and contracting (eg, endothelin) factors, and accumulated leukocytes produce a large amount of NO.^{43 44} The interactions between these factors mediate the vascular tone.⁴⁵ We have reported previously²¹ that the imbalance may contribute to remarkable vasoconstriction and subsequent vasodilation. In this study, tacrolimus administration caused vasoconstriction both arteries and veins throughout the experiment. Inactivation of neural NOS, as mentioned above,⁴ would partially contribute to vasoconstriction. In addition, tacrolimus treatment reduced the number of accumulated leukocytes,^{8 9} which produced a large amount of NO.^{43 44} Furthermore, tacrolimus has also been reported to suppress the production of the inducible NOS in cultured macrophages.⁴⁶

In conclusion, leukocyte recruitment to the inflammatory region is mediated through a multistep process.^{32 33} While accumulated leukocytes contribute to host protection, they are also involved in postischemic tissue injury.⁹ The present study demonstrated the inhibitory effect of tacrolimus on leukocyte rolling and accumulation and subsequent tissue injury. Our method will allow us to evaluate quantitatively the effectiveness of therapies involving leukocyte participation in the retina and will help us to estimate leukocyte involvement in neural damage in the brain after transient ischemia.

	Selected Abbreviations and Acronyms

 

AO = acridine orange GCL = ganglion cell layer IL = interleukin INL = inner nuclear layer IPL = inner plexiform layer NO = nitric oxide NOS = nitric oxide synthase ONL = outer nuclear layer

	Acknowledgments

 
This study was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture (Y.O., J.K., Y.H.) and a grant-in-aid for scientific research from the Ministry of Health and Welfare of Japan (J.K.).

Received February 2, 1998; revision received March 26, 1998; accepted April 15, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Sharkey J, Crawford JH, Butcher SP, Marston HM. Tacrolimus (FK506) ameliorates skilled motor deficits produced by middle cerebral artery occlusion in rats. Stroke. 1996;27:2282–2286.[Abstract/Free Full Text]

2. Sharkey J, Butcher SP. Immunophilins mediate the neuroprotective effects of FK506 in focal cerebral ischaemia. Nature. 1994;371:336–339.[Medline] [Order article via Infotrieve]

3. Yagita Y, Kitagawa K, Matsushita K, Taguchi A, Mabuchi T, Ohtsuki T, Yanagihara T, Matsumoto M. Effect of immunosuppressant FK506 on ischemia-induced degeneration of hippocampal neurons in gerbils. Life Sci. 1996;59:1643–1650.[Medline] [Order article via Infotrieve]

4. Dawson TM, Steiner JP, Dawson VL, Dinerman JL, Uhl GR, Snyder SH. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci U S A. 1993;90:9808–9812.[Abstract/Free Full Text]

5. Zhang J, Steiner JP. Nitric oxide synthase, immunophilins and poly(ADP-ribose) synthetase: novel targets for the development of neuroprotective drugs. Neurol Res. 1995;17:285–288.[Medline] [Order article via Infotrieve]

6. Lu CY, Sicher SC, Vazquez MA. Prevention and treatment of renal allograft rejection: new therapeutic approaches and new insights into established therapies. J Am Soc Nephrol. 1993;4:1239–1256.[Abstract]

7. Schreiber SL, Crabtree GR. The mechanism of action of cyclosporin A and FK506. Immunol Today. 1992;13:136–142.[Medline] [Order article via Infotrieve]

8. Wakabayashi H, Karasawa Y, Tanaka S, Kokudo Y, Maeba T. The effect of FK506 on warm ischemia and reperfusion injury in the rat liver. Surg Today. 1994;24:994–1002.[Medline] [Order article via Infotrieve]

9. Suzuki S, Toledo-Pereyra LH, Rodriguez FJ, Cejalvo D. Neutrophil infiltration as an important factor in liver ischemia and reperfusion injury: modulating effects of FK506 and cyclosporine. Transplantation. 1993;55:1265–1272.[Medline] [Order article via Infotrieve]

10. Zhang RL, Chopp M, Chen H, Garcia JH. Temporal profile of ischemic tissue damage, neutrophil response, and vascular plugging following permanent and transient (2H) middle cerebral artery occlusion in the rat. J Neurol Sci. 1994;125:3–10.[Medline] [Order article via Infotrieve]

11. Fukumura D, Salehi HA, Witwer B, Tuma RF, Melder RJ, Jain RK. Tumor necrosis factor alpha-induced leukocyte adhesion in normal and tumor vessels: effect of tumor type, transplantation site, and host strain. Cancer Res. 1995;55:4824–4829.[Abstract/Free Full Text]

12. Dirnagl U, Niwa K, Sixt G, Villringer A. Cortical hypoperfusion after global forebrain ischemia in rats is not caused by microvascular leukocyte plugging. Stroke. 1994;25:1028–1038.[Abstract]

13. Villringer A, Dirnagl U, Them A, Schurer L, Krombach F, Einhaupl KM. Imaging of leukocytes within the rat brain cortex in vivo. Microvasc Res. 1991;42:305–315.[Medline] [Order article via Infotrieve]

14. Distler C, Bronzel M, Paas I, Wahle P. Biochemical and histological analysis of two Muller cell antibodies in developing and adult cat and rat central nervous system. Cell Tissue Res. 1997;289:411–426.[Medline] [Order article via Infotrieve]

15. Duchini A, Govindarajan S, Santucci M, Zampi G, Hofman FM. Effects of tumor necrosis factor-alpha and interleukin-6 on fluid-phase permeability and ammonia diffusion in CNS-derived endothelial cells. J Investig Med. 1996;44:474–482.[Medline] [Order article via Infotrieve]

16. Greenwood J, Pryce G, Devine L, Male DK, dos-Santos WL, Calder VL, Adamson P. SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. J Neuroimmunol. 1996;71:51–63.[Medline] [Order article via Infotrieve]

17. Kimura H, Kiryu J, Nishiwaki H, Ogura Y. A new fluorescent imaging procedure in vivo for evaluation of the retinal microcirculation in rats. Curr Eye Res. 1995;14:223–228.[Medline] [Order article via Infotrieve]

18. Nishiwaki H, Ogura Y, Kimura H, Kiryu J, Honda Y. Quantitative evaluation of leukocyte dynamics in retinal microcirculation. Invest Ophthalmol Vis Sci. 1995;36:123–130.[Abstract/Free Full Text]

19. Nishiwaki H, Ogura Y, Kimura H, Kiryu J, Miyamoto K, Matsuda N. Visualization and quantitative analysis of leukocyte dynamics in retinal microcirculation of rats. Invest Ophthalmol Vis Sci. 1996;37:1341–1347.[Abstract/Free Full Text]

20. Miyamoto K, Ogura Y, Hamada M, Nishiwaki H, Hiroshiba N, Honda Y. In vivo quantification of leukocyte behavior in the retina during endotoxin-induced uveitis. Invest Ophthalmol Vis Sci. 1996;37:2708–2715.[Abstract/Free Full Text]

21. Tsujikawa A, Ogura Y, Hiroshiba N, Miyamoto K, Kiryu J, Honda. Y. In vivo evaluation of leukocyte dynamics in retinal ischemia reperfusion injury. Invest Ophthalmol Vis Sci. 1998;39:793–800.[Abstract/Free Full Text]

22. Stefansson E, Wilson CA, Schoen T, Kuwabara T. Experimental ischemia induces cell mitosis in the adult rat retina. Invest Ophthalmol Vis Sci. 1988;29:1050–1055.[Abstract/Free Full Text]

23. Hangai M, Yoshimura N, Yoshida M, Yabuuchi K, Honda Y. Interleukin-1 gene expression in transient retinal ischemia in the rat. Invest Ophthalmol Vis Sci. 1995;36:571–578.[Abstract/Free Full Text]

24. Darzynkiewicz Z, Kapuscinski J. Acridine orange: a versatile probe of nucleic acids and other cell constituents. In: Flow Cytometry and Sorting. New York, NY: Wiley-Liss Inc; 1990:219–314.

25. Hughes WF. Quantitation of ischemic damage in the rat retina. Exp Eye Res. 1991;53:573–582.[Medline] [Order article via Infotrieve]

26. Weber M, Mohand-Said S, Hicks D, Dreyfus H, Sahel JA. Monosialoganglioside GM1 reduces ischemia–reperfusion-induced injury in the rat retina. Invest Ophthalmol Vis Sci. 1996;37:267–273.[Abstract/Free Full Text]

27. Hayashi A, Weinberger AW, Kim HC, de Juan E Jr. Genistein, a protein tyrosine kinase inhibitor, ameliorates retinal degeneration after ischemia-reperfusion injury in rat. Invest Ophthalmol Vis Sci. 1997;38:1193–1202.[Abstract/Free Full Text]

28. del-Zoppo GJ, Schmid-Schonbein GW, Mori E, Copeland BR, Chang CM. Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons. Stroke. 1991;22:1276–1283.[Abstract/Free Full Text]

29. Heinel LA, Rubin S, Rosenwasser RH, Vasthare US, Tuma RF. Leukocyte involvement in cerebral infarct generation after ischemia and reperfusion. Brain Res Bull. 1994;34:137–141.[Medline] [Order article via Infotrieve]

30. Werns SW, Shea MJ, Lucchesi BR. Free radicals in ischemic myocardial injury. J Free Radic Biol Med. 1985;1:103–110.[Medline] [Order article via Infotrieve]

31. Ghezzi P, Dinarello CA, Bianchi M, Rosandich ME, Repine JE, White CW. Hypoxia increases production of interleukin-1 and tumor necrosis factor by human mononuclear cells. Cytokine. 1991;3:189–194.[Medline] [Order article via Infotrieve]

32. Bevilacqua MP, Nelson RM. Selectins. J Clin Invest. 1993;91:379–387.

33. Osborn L. Leukocyte adhesion to endothelium in inflammation. Cell. 1990;62:3–6.[Medline] [Order article via Infotrieve]

34. Strachan RD, Kane PJ, Cook S, Chambers IR, Clayton CB, Mendelow AD. Immunosuppression by whole-body irradiation and its effect on oedema in experimental cerebral ischaemia. Acta Neurol Scand. 1992;86:256–259.[Medline] [Order article via Infotrieve]

35. Liu J, Farmer J. J. D., Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807–815.[Medline] [Order article via Infotrieve]

36. Shibasaki F, McKeon F. Calcineurin functions in Ca(2+)-activated cell death in mammalian cells. J Cell Biol. 1995;131:735–743.[Abstract/Free Full Text]

37. Fruman DA, Mather PE, Burakoff SJ, Bierer BE. Correlation of calcineurin phosphatase activity and programmed cell death in murine T cell hybridomas. Eur J Immunol. 1992;22:2513–2517.[Medline] [Order article via Infotrieve]

38. Frantz B, Nordby EC, Bren G, Steffan N, Paya CV, Kincaid RL, Tocci MJ, O'Keefe SJ, O'Neill EA. Calcineurin acts in synergy with PMA to inactivate I B/MAD3, an inhibitor of NF-B. Embo J. 1994;13:861–870.[Medline] [Order article via Infotrieve]

39. Su MS, Semerijan A. Activation of transcription factor NF-B in Jurkat cells is inhibited selectively by FK506 in a signal-dependent manner. Transplant Proc. 1991;23:2912–2915.[Medline] [Order article via Infotrieve]

40. Yoshimoto T, Houkin K, Tada M, Abe H. Induction of cytokines, chemokines and adhesion molecule mRNA in a rat forebrain reperfusion model. Acta Neuropathol (Berl). 1997;93:154–158.[Medline] [Order article via Infotrieve]

41. Rothlein R, Czajkowski M, O'Neill MM, Marlin SD, Mainolfi E, Merluzzi VJ. Induction of intercellular adhesion molecule 1 on primary and continuous cell lines by pro-inflammatory cytokines: regulation by pharmacologic agents and neutralizing antibodies. J Immunol. 1988;141:1665–1669.[Abstract]

42. Graber N, Gopal TV, Wilson D, Beall LD, Polte T, Newman W. T cells bind to cytokine-activated endothelial cells via a novel, inducible sialoglycoprotein and endothelial leukocyte adhesion molecule-1. J Immunol. 1990;145:819–830.[Abstract]

43. Hangai M, Yoshimura N, Hiroi K, Mandai M, Honda Y. Inducible nitric oxide synthase in retinal ischemia-reperfusion injury. Exp Eye Res. 1996;63:501–509.[Medline] [Order article via Infotrieve]

44. Clark RS, Kochanek PM, Schwarz MA, Schiding JK, Turner DS, Chen M, Carlos TM, Watkins SC. Inducible nitric oxide synthase expression in cerebrovascular smooth muscle and neutrophils after traumatic brain injury in immature rats. Pediatr Res. 1996;39:784–790.[Medline] [Order article via Infotrieve]

45. Hiramatsu T, Forbess J, Miura T, Roth SJ, Cioffi MA, Mayer J Jr. Effects of endothelin-1 and endothelin-A receptor antagonist on recovery after hypothermic cardioplegic ischemia in neonatal lamb hearts. Circulation. 1995;92(suppl II):II-400-II-404.

46. Conde M, Andrade J, Bedoya FJ, Santa-Maria C, Sobrino F. Inhibitory effect of cyclosporin A and FK506 on nitric oxide production by cultured macrophages: evidence of a direct effect on nitric oxide synthase activity. Immunology. 1995;84:476–481.[Medline] [Order article via Infotrieve]

Editorial Comment

Valina L. Dawson, PhD, Guest Editor

Department Of Neurology Johns Hopkins University School Of Medicine Baltimore, Maryland

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
The immunosuppressant drugs cyclosporin A, FK506 (tacrolimus), and rapamyocin have revolutionized the field of organ transplantation. These agents bind to small proteins termed immunophilins.¹ For immune suppression, the drug-immunophilin complex inhibits the calcium-dependent serine/threonine protein phosphatase calcineurin. Inhibition of calcineurin prevents dephosphorylation of the nuclear factor of activated T cells, preventing its entry into the nucleus and thereby inhibiting initiation of interleukin-2 transcription.¹ Activation and growth of T cells is inhibited. In addition to inhibiting calcineurin activity, immunophilins have a wide range of intracellular protein targets that can mediate responses other than immunosuppression.

Recently, cyclosporin-A and FK506 have come to the attention of the neuroscience community because of their unique and powerful neuroprotective and neuroregenerative properties.^{2 3} FK506 has been shown to be neuroprotective in a variety of models of cerebral ischemia, including both focal and global ischemia, as well as transient and permanent ischemia. Cyclosporin has also been shown to be neuroprotective, although at higher doses than FK506. In vitro, in primary cortical cultures the neuroprotection provided by FK506 against excitotoxicity is mediated in part by preventing dephosphorylation and activation of neuronal NOS by calcineurin. It is proposed that FK506 is neuroprotective after cerebral ischemic insult through inhibition of the catalytic activity of neuronal NOS.^{2 3} However, recent studies⁴ show that FK506 does not protect against the excitotoxins quinolinate, NMDA, or AMPA when injected intraparenchamaly in vivo, suggesting that the mechanism of neuroprotection elicited by FK506 after cerebral ischemic insult may occur by mechanisms other than inhibition of neuronal NOS.

In the accompanying article, Tsujikawa and colleagues make a unique observation and present a novel hypothesis for a potential mechanism for the neuroprotection elicited by FK506. In the study the authors induce retinal ischemia by temporarily ligating the optic sheath. In animals treated with FK506, the total number of rolling leukocytes is markedly reduced. In addition, the velocity of rolling leukocytes is significantly faster in FK506-treated animals. The net result is a reduction of leukocyte accumulation following reperfusion. The reduction in leukocyte accumulation correlates with the degree of neuroprotection elicited by FK506 in this temporary ischemic insult model in the retina. These data suggest that one mechanism of FK506-mediated neuroprotection in cerebral ischemic injury is through its immunosuppressant activities on leukocyte activation and entry into postischemic tissue. FK506 has also been shown to suppress the induction of immunologic NOS in cultured macrophages. Iadecola and colleagues⁵ have shown that immunologic NOS induction plays a critical role in late-stage neurological damage following ischemic insult. Therefore, the actions of FK506 in suppressing multiple components of the immune system may contribute significantly to its neuroprotective effects after cerebral ischemic insult. These data confirm and extend the usefulness of FK506 or like agents, for potential therapy in the treatment of stroke.

	Selected Abbreviations and Acronyms

 

AO = acridine orange GCL = ganglion cell layer IL = interleukin INL = inner nuclear layer IPL = inner plexiform layer NO = nitric oxide NOS = nitric oxide synthase ONL = outer nuclear layer

Received February 2, 1998; revision received March 26, 1998; accepted April 15, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
1. Schreiber SL. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science. 1991; 251:283–287.

2. Dawson TM. Immunosuppressants, immunophilins, and the nervous system. Ann Neurol. 1996; 40:559–560.

3. Snyder SH, Sabatini DM, Lai MM, Steiner JP, Hamilton GS, Suzdak PD. Neural actions of immunophilin ligands. Trends Pharmacol Sci. 1998; 19:21–26.

4. Butcher SP, Henshall DC, Teramura Y, Iwasaki K, Sharkey J. Neuroprotective actions of FK506 in experimental stroke: in vivo evidence against an antiexcitotoxic mechanism. J Neurosci. 1997; 17:6939–6946.

5. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends Neurosci. 1997; 20:132–139.

This article has been cited by other articles:

				N. Jo, G.-S. Wu, and N. A. Rao Upregulation of Chemokine Expression in the Retinal Vasculature in Ischemia-Reperfusion Injury Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 4054 - 4060. [Abstract] [Full Text] [PDF]

				S. Miyahara, J. Kiryu, A. Tsujikawa, H. Katsuta, K. Nishijima, K. Miyamoto, K. Yamashiro, A. Nonaka, and Y. Honda Argatroban Attenuates Leukocyte- and Platelet-Endothelial Cell Interactions After Transient Retinal Ischemia Stroke, August 1, 2003; 34(8): 2043 - 2049. [Abstract] [Full Text] [PDF]

				S. Kaja, S.-H. Yang, J. Wei, K. Fujitani, R. Liu, A.-M. Brun-Zinkernagel, J. W. Simpkins, K. Inokuchi, and P. Koulen Estrogen Protects the Inner Retina from Apoptosis and Ischemia-Induced Loss of Vesl-1L/Homer 1c Immunoreactive Synaptic Connections Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 3155 - 3162. [Abstract] [Full Text] [PDF]

				M. Honjo, H. Tanihara, K. Nishijima, J. Kiryu, Y. Honda, B. Y. J. T. Yue, and T. Sawamura Statin Inhibits Leukocyte-Endothelial Interaction and Prevents Neuronal Death Induced by Ischemia-Reperfusion Injury in the Rat Retina Arch Ophthalmol, December 1, 2002; 120(12): 1707 - 1713. [Abstract] [Full Text] [PDF]

				A. Nonaka, J. Kiryu, A. Tsujikawa, K. Yamashiro, K. Nishijima, K. Miyamoto, H. Nishiwaki, Y. Honda, and Y. Ogura Inhibitory Effect of Ischemic Preconditioning on Leukocyte Participation in Retinal Ischemia-Reperfusion Injury Invest. Ophthalmol. Vis. Sci., September 1, 2001; 42(10): 2380 - 2385. [Abstract] [Full Text] [PDF]

				L. Lang-Lazdunski, C. Heurteaux, H. Dupont, D. Rouelle, C. Widmann, and J. Mantz The Effects of FK506 on Neurologic and Histopathologic Outcome After Transient Spinal Cord Ischemia Induced by Aortic Cross-Clamping in Rats Anesth. Analg., May 1, 2001; 92(5): 1237 - 1244. [Abstract] [Full Text] [PDF]

				A. Tsujikawa, J. Kiryu, A. Nonaka, K. Yamashiro, H. Nishiwaki, Y. Honda, and Y. Ogura Leukocyte-endothelial cell interactions in diabetic retina after transient retinal ischemia Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2000; 279(3): R980 - R989. [Abstract] [Full Text] [PDF]

				A. Nonaka, J. Kiryu, A. Tsujikawa, K. Yamashiro, K. Miyamoto, H. Nishiwaki, M. Mandai, Y. Honda, and Y. Ogura Administration of 17{beta}-Estradiol Attenuates Retinal Ischemia-Reperfusion Injury in Rats Invest. Ophthalmol. Vis. Sci., August 1, 2000; 41(9): 2689 - 2696. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tsujikawa, A. Articles by Dawson, V. L. Search for Related Content PubMed PubMed Citation Articles by Tsujikawa, A. Articles by Dawson, V. L.