November 2009
Volume 50, Issue 11
Free
Visual Psychophysics and Physiological Optics  |   November 2009
The Porcine Retinal Vasculature Accessed Using an Endovascular Approach: A New Experimental Model for Retinal Ischemia
Author Affiliations & Notes
  • Håkan Morén
    From the Departments of Ophthalmology,
  • Per Undrén
    Neuroradiology, and
  • Bodil Gesslein
    From the Departments of Ophthalmology,
  • Göran K. Olivecrona
    Cardiology, Lund University, Lund, Sweden.
  • Sten Andreasson
    From the Departments of Ophthalmology,
  • Malin Malmsjö
    From the Departments of Ophthalmology,
  • Corresponding author: Malin Malmsjö, Department of Ophthalmology, Lund University, BMC A13, SE-221 84 Lund, Sweden; malin.malmsjo@med.lu.se
Investigative Ophthalmology & Visual Science November 2009, Vol.50, 5504-5510. doi:10.1167/iovs.09-3529
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      Håkan Morén, Per Undrén, Bodil Gesslein, Göran K. Olivecrona, Sten Andreasson, Malin Malmsjö; The Porcine Retinal Vasculature Accessed Using an Endovascular Approach: A New Experimental Model for Retinal Ischemia. Invest. Ophthalmol. Vis. Sci. 2009;50(11):5504-5510. doi: 10.1167/iovs.09-3529.

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

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Abstract

Purpose.: The aim of this study was to examine whether the retinal circulation in the pig can be accessed using interventional neuroradiology and to explore the possibility of creating occlusions that result in experimental retinal ischemia.

Methods.: Six experiments were performed using 100-kg pigs. The external carotid system was catheterized using a fluoroscopy-monitored, transfemoral, endovascular approach. Transient and permanent vascular occlusions were performed using an angioplasty balloon catheter or a liquid embolic agent that was administered via an injection-catheter.

Results.: A technique for transfemoral catheterization of arteries supplying the retina was established. The ophthalmic artery was demonstrated to give rise to the main ciliary artery from which the retinal artery branched as a single artery or as several arteries. A balloon-catheter could be introduced into the ophthalmic artery but not into the main ciliary artery. An injection-catheter could, in all experiments, be introduced into the main ciliary artery and, in some experiments, into the retinal artery. Occlusion of the ophthalmic artery, over the branching of the main ciliary artery, caused incomplete ischemia, presumably because of collaterals feeding the distal parts of the vasculature. Multifocal ERG (mfERG) recordings showed decreased amplitudes and increased implicit times, indicating retinal ischemia. Occlusion of the ciliary and retinal arteries caused complete ischemia, as shown by complete flattening of the mfERG recordings and, by indirect ophthalmoscopy, blanching of the retinal arteries and a pale retina

Conclusions.: The authors show for the first time that the ophthalmic and retinal artery can be catheterized using a transfemoral endovascular approach. This technique may be useful to produce clear-cut experimental retinal ischemia.

A number of animal models have been developed to study retinal ischemia, although many of these have limitations. Ischemia, by elevating the intraocular pressure, is a frequently used model that produces global ischemia identical with that seen in central retinal artery occlusion (CRAO). 13 However, the retinal injury may result from both ischemia and pressure. Vascular ligation is another common method of causing retinal ischemia and is achieved, in its simplest form, by placing a suture around the optic nerve bundle. 4 This occludes blood flow, elevates the intraocular pressure (caused by pressure on the globe), and constricts the optic nerves, which damages axons. 3 Ligating the posterior ciliary vessels independently of the optic nerve can be performed in rats, although it is technically more demanding. This produces features similar to those seen after CRAO but also causes uveal ischemia. Incomplete ischemia can be produced by ligating more proximal arteries in the neck, and the degree of ischemia that is produced depends of the number of vessels ligated. 58 This intervention may mimic carotid insufficiency, but the principal problem is that optic nerve ischemia and cerebral infarction are also produced. 8 Retinal vessels can be occluded by intravenous injection of rose bengal, a photosensitive dye, followed by intense retinal illumination, resulting in thrombosis of retinal vessels that can mimic branch retinal artery occlusion (BRAO), 911 although there may be retinal injury secondary to phototoxicity in addition to an ischemic component. BRAO may also be produced by laser photocoagulation or transvitreal diathermia, 12,13 but theses measures may have direct effects on the retina with, for example, ruptures of Bruch's membrane. 
In conclusion, many of the commonly used animal models for retinal ischemia have limitations. 3 The clinical relevance of any data in the laboratory depends on the nature of extrapolation. If an experimental model of retinal ischemia can replicate human pathology and pharmacological treatments can ameliorate the induced pathology, then it is a logical assumption that such treatments may be effective in the clinic and merit further investigation. Clearly, the ability to extrapolate data from an animal model to the clinical situation requires an experimental model that closely resembles retinal ischemia in humans. The retinal anatomy of pigs is virtually identical to that of humans. 14 The porcine eye appears to have a typical primate-like architecture and is similar to the human eye regarding both size and retinal blood supply. 14 The pig has also proven to be a suitable animal for experimental analysis of the retina and retinal arteries. 15 If clear-cut retinal ischemia, without limiting confounding factors, can be created in the pig, this may become an experimental model that closely resembles pathology in humans. 
We hypothesized that one way to create a clear-cut experimental model of retinal ischemia may be by accessing the retinal circulation by transfemoral endovascular catheterization. The porcine internal cerebral circulation is not accessible for catheterization because of a rete mirabilis, and the pig has therefore been regarded as unsuitable for cerebral infarction projects. 16,17 However, in the pig, the ophthalmic artery derives from the external carotid circulation. The external carotid artery gives off the maxillary artery from which the ophthalmic artery branches. 18,19 In 1992, Scheurer et al. 1921 managed to catheterize the external maxillary artery and inject microparticles before the branching of the ophthalmic artery. However, because the injections were made in the maxillary artery, which is a large artery supplying major parts of the head, ischemia was presumably not produced only in the retina. Selective endovascular catheterization of the ophthalmic or retinal arteries has to our knowledge never been performed before in any animal model. 
The aim of the present study was to explore the retinal circulation in the pig using a catheter-based transfemoral endovascular approach and to examine the possibilities of creating experimental retinal ischemia with this technique. We therefore, for the first time, present a method for catheterization of the ophthalmic, main ciliary, and retinal arteries in the pig. Furthermore, different occlusion techniques, including balloon catheter and liquid embolic agent, are explored. The effects of occlusion on the retina are examined using multifocal electroretinogram (mfERG) and indirect ophthalmoscopy. 
Materials and Methods
Animals and Anesthesia
Domestic Landrace pigs of both sexes, with a mean body weight of 100 kg, were used for the study. Before the surgical procedure, the animals were fasted overnight with free access to water. An intramuscular injection of 15 mg/kg body weight ketamine (Ketaminol vet, 100 mg/mL; Farmaceutici Gellini S.p.A, Aprilia, Italy) in combination with 2 mg/kg xylazine (Rompun vet, 20 mg/mL; Bayer AG, Leverkusen, Germany) was used for premedication. Anesthesia was maintained with 1 to 2 mL thiopental (Pentothal; 50 mg/mL, when necessary; Abbott, Stockholm, Sweden), in combination with fentanyl (Leptanal; 0.02 μg/kg body weight). Mechanical ventilation was established with a ventilator (900B; Siemens-Elema, Solna, Sweden) in the volume-controlled mode. Continuous monitoring of the animal was performed using electrocardiogram and arterial pH, pO2, and pCO2, during the angiographic procedure. After the experiment, the animals were euthanatized by an overdose of potassium. 
Six catheterization experiments were performed. A 6-F vascular sheath was inserted in the right femoral artery, using a percutaneous approach (Radiofocus Introducer II, 6 F; Terumo Europe N.V., Leuven, Belgium). A 5-F angiographic catheter (Tempo 5; Headhunter Catheter, Cordis, South Ascot, UK) was then inserted via the external carotid artery into the maxillary artery using fluoroscopic guidance. A cerebral angiography of the external carotid system was then performed, and sagittal and coronal road-map images were obtained for guiding of the procedure. The 5-F active tracking catheter was then exchanged for a 6-F guide catheter (Envoy Guide catheter, 6 F; Cordis), which was inserted into the external carotid artery. Using the 6-F guide catheter, two different approaches to perform occlusion of the retinal arterial circulation were performed. 
A 4-F balloon catheter (Hyperglide Occlusion Balloon, 4 × 10 mm, ev3; Neurovascular, Irvine, CA) was advanced over a 0.010-inch guidewire (X-pedion Guidewire, 0.010 inch, 200 cm, ev3; Neurovascular). Occlusion of the main ciliary artery was achieved by inflating the balloon in the ophthalmic artery at the place for the branching of the main ciliary artery. 
A 1.5-F injection catheter (Marathon Flow Directed Micro Catheter, 1.5 F, or UltraFlow Flow Directed Micro Catheter, 1.5 F, ev3; Neurovascular) was advanced over a 0.008-inch guidewire (Mirage Guidewire, ev3; Neurovascular). A nonadhesive liquid embolic agent (Onyx HD-500, ev3; Neurovascular) was used for occlusion. 
The nonadhesive liquid embolic agent (Onyx HD-500, ev3; Neurovascular) is used for embolization of brain arteriovenous malformations. It has a slow, controlled injection and delivery method. In addition, it has the properties of a substance that is modulated to adapt the shape of the injection site but does not embolize or disappear from the site of injection. The nonadhesive liquid embolic agent (Onyx HD-500, ev3; Neurovascular) was injected in two different locations: (1) at the place for the branching of the main ciliary artery and (2) more distally into the ciliary artery and the branching retinal arteries. Eyes that were subject to injection were dissected and photographed after termination of the experiments for analysis of the placement of the liquid embolic agent under a microscope. 
Evaluation of the Retina
The eyes were dilated with topical cyclopentolate hydrochloride (Cyclogyl 1%; Alcon Laboratories, Inc., Fort Worth, TX). Indirect ophthalmoscopy was performed for macroscopic evaluation of the retina, in which blanching of the retinal arteries and a pale retina suggested ischemia. mfERG was performed before and after endovascular artery occlusion for evaluation of retinal function, as described. After termination of the experiments, the eyes were dissected and examined under a microscope. 
Multifocal ERG (mfERG)
The animals were kept in normal room light for 1 hour before stimulation and during stimulation. For pupil dilatation, the eyes were dilated with topical cyclopentolate hydrochloride (1%; Cyclogyl) to a pupil diameter of 8 to 10 mm. A Burian-Allen bipolar contact lens electrode with built-in infrared emitters (Hansen Ophthalmic Development Laboratory; Iowa City, Iowa, IA) lubricated with 2% hydroxypropyl-methylcellulose (Methocel; Dow Chemical, Midland, MI) was applied to the eye, and a ground electrode needle was inserted into the skin behind the ear. The recordings were made using a visual evoked response imaging system (VERIS Science 4.3; EDI, San Mateo, CA). The stimulus, consisting of a picture with 103 geometric patches (unscaled hexagons) was delivered by a miniature cathode ray tube. The pattern seemed to flicker randomly, but each element followed a fixed, predetermined sequence (the m-sequence). The equipment was appropriately calibrated according to the instructions and devices from the manufacturers regarding both the grid and the luminance. The light intensity in the recording area was 0.110 lux. In every m-sequence, two additional blank, dark frames were inserted. The signal gain was 100,000, and the filter range 3 to 300 Hz with no additional notch filtering. Luminance flickered between light and dark according to a pseudorandom binary m-sequence of 75 Hz, with a mean stimulus luminance of 16.6 cd/m2 and a flash intensity of 1.33 cd · s/m2. Spatial averaging was set to 17%, as in the settings for the Veris Clinic. One iteration of the artifact rejection system included in the VERIS software was used. 
By means of an infrared camera, the fundus was visualized with infrared light from the recording electrode, allowing continual visualization of the retina during the examination, and the stimulus pattern was consistently positioned with the optic nerve head at the lower central part of the recording area. 
The mfERG traces were analyzed according to the different areas of the central part of the retina for monitoring of localized alterations. Area 1 corresponds to the visual streak, area 2 corresponds to the area between the visual streak and the optic nerve head, and area 3 corresponds to the area around the optic nerve head. 
Statistical Analysis
Statistical analysis was performed using Student's t-test. Statistical analysis was performed using the Mann-Whitney U test. Results are presented as mean ± SEM. 
Ethics
All proceedings and animal treatment were conducted in accordance with the guidelines of the Ethics Committee of Lund University, the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication no. 85–23, revised 1985), and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Results
Vascular Anatomy
Angiography of the carotid system revealed a truncus bicaroticus that split into a left and a right common carotid artery. There was faint internal but prominent external carotid circulation. The common carotid artery gives off, as a small side branch, the ascending pharyngeal artery, which is the equivalent of the internal carotid artery in humans. The ascending pharyngeal artery divides into tiny infrabasal arteries that anastomose into an arteriolar network at the skull base, the rete mirabilis. The rete mirabilis prevents any catheterization of the intracerebral arterial territory in the pig. 
The external carotid artery seemed to continue from the common carotid artery, and this vascular system was easily accessed by catheterization. The maxillary artery branches from the external carotid artery. The maxillary artery gives off the temporal, the lingual, the auricular, the facial, and the buccinator artery. The maxillary artery gives rise to the infraorbital artery. The ophthalmic artery branches of the infraorbital artery. After having accessed the ophthalmic artery, injection of contrast produced a characteristic half-moon–shaped outline of the retina. The ophthalmic artery gives off the main ciliary artery, from which the retinal artery branches as a single artery or several arteries. For angiograms, see Figure 1
Figure 1.
 
Angiogram of the left common carotid artery in a pig. (A) Basal view. (B) Lateral view. (C) Enlargement of the inset in (B). The (1) ascending pharyngeal artery originates, as a small side branch, from the (2) common carotid artery and feeds the (3) rete mirabilis, which then converges to form the intracranial carotid artery. The (4) external carotid artery is a continuity of the common carotid artery. The (5) maxillary artery branches from the external carotid artery. The maxillary artery gives off the (6) lingual, the (7) auricular, the (8) facial, and the (9) buccinator arteries. The maxillary artery gives rise to the (10) infraorbital artery. The (11) ophthalmic artery branches off the infraorbital artery. After having accessed the ophthalmic artery, injection of contrast will produce a characteristic half-moon–shaped outline of the retina (arrows). The ophthalmic artery gives off the main ciliary artery, (12) from which the retinal artery branches.
Figure 1.
 
Angiogram of the left common carotid artery in a pig. (A) Basal view. (B) Lateral view. (C) Enlargement of the inset in (B). The (1) ascending pharyngeal artery originates, as a small side branch, from the (2) common carotid artery and feeds the (3) rete mirabilis, which then converges to form the intracranial carotid artery. The (4) external carotid artery is a continuity of the common carotid artery. The (5) maxillary artery branches from the external carotid artery. The maxillary artery gives off the (6) lingual, the (7) auricular, the (8) facial, and the (9) buccinator arteries. The maxillary artery gives rise to the (10) infraorbital artery. The (11) ophthalmic artery branches off the infraorbital artery. After having accessed the ophthalmic artery, injection of contrast will produce a characteristic half-moon–shaped outline of the retina (arrows). The ophthalmic artery gives off the main ciliary artery, (12) from which the retinal artery branches.
A balloon catheter could be introduced into the ophthalmic artery but not into the main ciliary artery (Fig. 2). An injection catheter is smaller in diameter than a balloon catheter and could, in all experiments, be introduced into the main ciliary artery and, in some experiments, into the retinal artery (Fig. 3). 
Figure 2.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). (B) Positioning of a balloon-catheter (arrow) in the ophthalmic artery (asterisk), with its tip at the branching of the main ciliary artery. (C) Angiogram during balloon occlusion of the ophthalmic artery. Note that contrast does not enter the ophthalmic artery during balloon occlusion.
Figure 2.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). (B) Positioning of a balloon-catheter (arrow) in the ophthalmic artery (asterisk), with its tip at the branching of the main ciliary artery. (C) Angiogram during balloon occlusion of the ophthalmic artery. Note that contrast does not enter the ophthalmic artery during balloon occlusion.
Figure 3.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). Both the ophthalmic artery (asterisk) and the main ciliary artery, with the branching of the retinal artery (arrow), are visible. Right: enlargement of the inset in the left panel, showing the main ciliary artery and the branching of the retinal artery (arrow). (B) Positioning of the catheter for injection of liquid embolic agent (arrow) in the main ciliary artery. Asterisk: ophthalmic artery. (C) Result after injection of the radiopaque liquid embolic agent in the main ciliary artery and the retinal artery (arrow). (D) Angiogram during liquid embolic agent occlusion of the main ciliary and retinal artery. Note that contrast does not enter the ophthalmic or main ciliary artery.
Figure 3.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). Both the ophthalmic artery (asterisk) and the main ciliary artery, with the branching of the retinal artery (arrow), are visible. Right: enlargement of the inset in the left panel, showing the main ciliary artery and the branching of the retinal artery (arrow). (B) Positioning of the catheter for injection of liquid embolic agent (arrow) in the main ciliary artery. Asterisk: ophthalmic artery. (C) Result after injection of the radiopaque liquid embolic agent in the main ciliary artery and the retinal artery (arrow). (D) Angiogram during liquid embolic agent occlusion of the main ciliary and retinal artery. Note that contrast does not enter the ophthalmic or main ciliary artery.
Occlusion Using a Balloon Catheter
For temporary occlusion, a balloon catheter was inflated for 1 hour in the ophthalmic artery, proximally, over the branching of the main ciliary artery. This occlusion hinders blood supply to the whole vascular system, including the ciliary and retinal artery branches that supply the retina. Occlusion of the ophthalmic artery did not result in any ophthalmologically inspectable signs of retinal ischemia, presumably because of collaterals feeding the distal parts of the vasculature. However, the mfERG recordings showed increased implicit times (20.91 ± 0.20 before and 21.67 ± 0.31 after occlusion [P < 0.05] in the area of the retina corresponding to the visual streak), and the amplitudes seemed to decrease (13.18 ± 2.45 before and 11.32 ±1.35 after ischemia [P = NS] in the visual streak area; Fig. 4), indicating incomplete ischemia. 
Figure 4.
 
Representative examples of the mfERG recordings. Left: topographical maps in which the optic nerve head (large arrows) and visual streak (small arrows) can be visualized. Right: individual recordings. mfERG recordings were obtained (A) before catheterization, (B) during balloon-occlusion of the ophthalmic artery, over the branching of the main ciliary artery, and (C) after injection of liquid embolic agent into the main ciliary and retinal arteries. Note that occlusion of the ophthalmic artery causes incomplete retinal ischemia with decreased amplitude and increased implicit time. Occlusion of the ciliary and retinal artery causes complete retinal ischemia with flattening of the mfERG traces.
Figure 4.
 
Representative examples of the mfERG recordings. Left: topographical maps in which the optic nerve head (large arrows) and visual streak (small arrows) can be visualized. Right: individual recordings. mfERG recordings were obtained (A) before catheterization, (B) during balloon-occlusion of the ophthalmic artery, over the branching of the main ciliary artery, and (C) after injection of liquid embolic agent into the main ciliary and retinal arteries. Note that occlusion of the ophthalmic artery causes incomplete retinal ischemia with decreased amplitude and increased implicit time. Occlusion of the ciliary and retinal artery causes complete retinal ischemia with flattening of the mfERG traces.
Occlusion Experiments Using a Liquid Embolic Agent
Liquid Embolic Agent in the Ophthalmic Artery.
For permanent occlusion, liquid embolic agent was injected in the ophthalmic artery, proximally, over the branching of the main ciliary artery. This occlusion hinders blood supply to the whole vascular system, including the ciliary and retinal artery branches that supply the retina. This occlusion did not result in any inspectable signs of retinal ischemia, whereas mfERG recordings showed increased implicit times (20.91 ± 0.20 before and 21.67 ± 0.31 after occlusion, in the area of the retina corresponding to the visual streak) and decreased amplitudes (13.18 ± 2.45 before and 11.32 ±1.35 after ischemia, in the visual streak area), suggesting incomplete retinal ischemia. 
Liquid Embolic Agent in the Main Ciliary Artery and in the Retinal Arteries.
An injection catheter could be introduced all the way into the main ciliary artery, which then branches into the ciliary and retinal arteries. From this site, liquid embolic agent was injected distally, into the ciliary artery and into the branching retinal arteries. This resulted in complete occlusion of the retinal circulation with ophthalmologically inspectable blanching of the retinal arteries and a pale retina. Furthermore, the waveforms of the mfERG were completely abolished (Fig. 4). Dissection of these eyes showed ciliary arteries that were filled with the liquid embolic agent (Fig. 5). 
Figure 5.
 
Photographs from a porcine eye in which liquid embolic agent was injected extensively in the ciliary and retinal arteries via transfemoral catheterization. (A) Photograph of the posterior portion of the dissected eye. The ciliary arteries are filled with the black liquid embolic agent. (B) Photograph of the eyecup with the retina, optic nerve head, and arteries. The retinal arteries are pale with no glue and are sparse with blood segmented in the vessels. (C) Angiogram of the same eye showing the ophthalmic artery (asterisk) and the ciliary and retinal arteries, all which are filled with the radiopaque liquid embolic agent (arrow).
Figure 5.
 
Photographs from a porcine eye in which liquid embolic agent was injected extensively in the ciliary and retinal arteries via transfemoral catheterization. (A) Photograph of the posterior portion of the dissected eye. The ciliary arteries are filled with the black liquid embolic agent. (B) Photograph of the eyecup with the retina, optic nerve head, and arteries. The retinal arteries are pale with no glue and are sparse with blood segmented in the vessels. (C) Angiogram of the same eye showing the ophthalmic artery (asterisk) and the ciliary and retinal arteries, all which are filled with the radiopaque liquid embolic agent (arrow).
Discussion
In the present study, we have identified the pathway for transfemoral endovascular catheterization of the vasculature that supplies the retina in the pig. We present a method for permanent and temporary occlusion of the ophthalmic artery and the branching ciliary and retinal arteries. The present study proves advances in interventional radiology for eye research that opens up new possibilities for developing experimental animal models for retinal ischemia. 
Catheterization of the Vasculature That Supplies the Retina
Detailed knowledge of the porcine vascular anatomy is a prerequisite for proper use of pigs as experimental models in interventional radiology. We have, in the present study, characterized the vascular structures to reach the retinal circulation. It has been shown by others that the cerebrovascular anatomy of domestic pigs differs from that of humans. 22 Pigs do not have an internal carotid artery. The common carotid artery supplies, as a small side branch, the ascending pharyngeal artery, which gives rise to the rete mirabilis—a network of fine arterioles resembling the gross morphology of a high-flow vascular malformation—at the cranial base. 18 The rete mirabilis then converges to form the intracranial carotid artery, which provides the major cerebral blood supply. The rete mirabilis cannot be passed by a selective angiographic catheter, 16,18 and it has been disappointing that the carotid circulation of the pig is not appropriate for cerebral infarction research. 17  
On the other hand, the pig has an extensive external carotid system that can be catheterized. 1921 The external carotid artery is a continuity of the common carotid artery. It supplies the lingual artery, the external maxillary artery, the auricular artery, the superficial temporal artery, the transverse facial artery, the internal maxillary artery, and branches to the parotid gland. 18 The large internal maxillary artery gives rise to the medial meningeal artery, the deep temporal artery, the mandibular artery, the buccinator artery, the ophthalmic artery, the malar artery, the nasal artery, the palatine artery, and the infraorbital artery. 18 Scheurer et al. 1921 managed to catheterize the maxillary artery in an attempt to cause retinal ischemia by injection of microparticles before the branching of the ophthalmic artery. However, the ophthalmic artery was never accessed in these studies. We show, for the first time, that the ophthalmic artery can be catheterized by a transfemoral endovascular approach via the external carotid and maxillary artery. In the present study, injection of contrast in the ophthalmic artery produced a characteristic half-moon–shaped outline of the retina. The ophthalmic artery was demonstrated to give rise to the main ciliary artery from which the retinal artery branched as a single or several arteries. Similar findings have been reported in light and electron microscopy studies. 23 In addition to the ophthalmic artery, we showed that the main ciliary artery and sometimes the retinal artery could also be catheterized. 
Occlusion of the Vasculature That Supplies the Retina
We believe that endovascular access to the retinal circulation is useful for creating experimental animal models for retinal ischemia. Experimental animal models for retinal ischemia, without limitations and confounding factors, are scarce. 3 One major advantage with occlusion of the retinal circulation using a transfemoral endovascular approach is that it affects only the blood supply and presumably does not have any unwanted side effects on the eye, such as pressure in high-IOP ischemia models or nerve effects in optic nerve bundle ligation ischemia models. 3  
In the present study, different approaches were used to cause occlusion of the blood supply to the retina. Proximal occlusion of the ophthalmic artery, over the branching of the main ciliary artery (which then supplies the ciliary and retinal artery branches), was achieved using a balloon catheter or injection of a liquid embolic agent. The balloon was used to achieve temporary artery occlusion, after which reperfusion may be allowed, whereas the liquid embolic agent was used to achieve permanent occlusion. Either of these procedures, in the ophthalmic artery, did not result in any ophthalmologically inspectable signs of retinal ischemia. However, the mfERG recordings showed decreased amplitudes and increased implicit times, which reflect retinal function as in partial ischemic injury. 2427 Presumably, such proximal occlusion allows collateral blood supply to the retina via anastomoses from, for example, the lingual artery, which may rescue the retina from complete ischemia and result in only partial ischemia. To overrule the contribution of blood flow from collaterals and to achieve total occlusion of retinal blood flow, the liquid embolic agent was injected more distally, into the ciliary and retinal arteries. This resulted in blanching of the retinal arteries and a pale retina, as observed with indirect ophthalmoscopy. Furthermore, the waveforms of the mfERG were abolished, suggesting complete ischemia. 
Taken together, the degree of ischemia depends on the proximity of the vascular occlusion to the retina. The probable reason for this is that collaterals anastomose to the distal parts of the vasculature. Distal occlusion thus results in a more extensive ischemia than proximal occlusion. Occlusion of the retinal arteries causes ophthalmoscopically evident complete retinal ischemia, whereas occlusions further from the retina (e.g., in the proximal parts of the main ciliary artery or in the ophthalmic artery) cause incomplete or partial ischemia. Using endovascular catheterization for creating retinal ischemia provides unique possibilities to elaborate with the degree of ischemia for creating an optimal experimental model of retinal ischemia. 
The Pig as an Experimental Model for Retinal Ischemia
There are limitations to the use of pigs in experimental setups for eye research. These are large animal experiments with all their implications; the experimental equipment, including a radiologic laboratory, is expensive, and gathering great numbers for statistical comparisons is demanding. On the other hand, using the pig for developing an experimental model for retinal ischemia may be beneficial for the following reasons. The ability to extrapolate data from an animal model to the clinical situation requires a model that closely resembles retinal ischemia in humans. There are anatomic similarities between the human and the porcine eye, macroscopically as well as histologically, 14 making this animal a good model when testing ophthalmological treatment modalities. Smaller laboratory animals, such as the rat, rabbit, and guinea pig, have commonly been used for creating experimental retinal ischemia, but one limiting factor has been the wide variety of retinal vascular patterns across species. The pig shares virtually identical retinal vascular anatomy with humans. 14  
The present technique for creating experimental retinal ischemia in pigs may in future be performed as recovery experiments for the evaluation of long-term effects of ischemic injury. Recovery experiments in pigs using transfemoral catheterization of the carotid circulation are feasible and have been demonstrated in numerous studies. 18,22 The femoral artery can be localized by palpation and then punctured transcutaneously. The advantage with this technique is that, after finalizing the catheterization experiments, groin hemostasis can be achieved and the animals can recover without incident. 17  
The overall aim is to develop a therapeutic strategy for retinal ischemia using this porcine model. If an experimental model of retinal ischemia can replicate human pathology, in which neovascularizations are crucial, and if pharmacologic treatments can ameliorate the induced pathology, then it is logically consistent to suggest that such treatment may be effective in the clinic. The next step, after the present study, will be to induce ischemia in the pig by interventional radiology, as described here, let the animals recover from anesthesia, and then hope that neovascularizations develop. The eyes can be examined repeatedly in vivo by ophthalmologic inspection, fluorescein angiography, OCT, and mfERG, and they can be analyzed further using molecular biological techniques, including real-time PCR, Western blot analysis, and immunofluorescence staining. We believe that the induction of angiogenesis is probable because experimental ischemia created by selective occlusion of the blood supply of the retina is similar to the pathology of retinal ischemia in humans and the porcine eye and its retinal vasculature are similar to those in humans. 14 However, this porcine experimental model of retinal ischemia must be further developed so that the degree of ischemia is titrated for optimal induction of neovascularization. This can be done when using interventional radiology because vascular occlusions can be produced at different levels in the vascular tree (i.e., a proximal occlusion creates partial ischemia, whereas a distal occlusion creates complete ischemia). When establishing a porcine model of retinal ischemia that results in neovascularizations, the pathogenesis of circulatory failure can be explored to identify new pharmacologic targets and inhibitors of angiogenesis. One way would be to induce ischemia with subsequent neovascularizations. After the induction of ischemia, one eye can be treated with an intravitreal or subconjunctival injection of a pharmacologic agent. The eyes can thereafter be examined with regard to attenuated ischemic retinal injury and angiogenesis 
Pig Size
Large animal size (100 kg, as used in the present study) is presumably a prerequisite to manage to catheterize these small arteries, including the main ciliary and retinal arteries. In our hands, the present experiments would be difficult in smaller pigs with finer vasculature. 
Conclusions
In the present study, we established a unique technique for transfemoral endovascular catheterization of the ophthalmic and retinal arteries. In this porcine model, the ophthalmic artery was demonstrated to give rise to the main ciliary artery, from which the retinal artery branched as a single artery or several arteries. Temporary occlusion of the ophthalmic artery at the branching of the main ciliary artery was produced by inflating a balloon. Permanent occlusion could be produced, both at the level of the ophthalmic artery at the branching of the main ciliary arteries, and secondly more distally in the ciliary and retinal arteries, using a liquid embolic agent. Distal occlusions resulted in complete ischemia, whereas proximal occlusions resulted in incomplete or partial ischemia presumably because of collaterals feeding the distal parts of the vasculature. Artery occlusion was verified using mfERG and indirect ophthalmoscopy. Endovascular catheterization may be a useful tool to produce clear-cut experimental retinal ischemia without confounding factors. Using the pig for performing these procedures may be beneficial because there are anatomic similarities between the human and the porcine eye, which facilitates extrapolation of the results to the clinical situation. 
Footnotes
 Supported by the Swedish Medical Research Council, Lund University Faculty of Medicine, a Swedish Government Grant for Clinical Research, Lund University Hospital Research Grants, the Swedish Medical Association, the Royal Physiographic Society in Lund, the Åke Wiberg Foundation, the Anders Otto Swärd Foundation/Ulrika Eklund Foundation, the Magn Bergvall Foundation, the Crafoord Foundation, the Anna-Lisa and Sven-Erik Nilsson Foundation, the Jeanssons Foundation, Kronprinsessan Margaretas Arbetsnämnd för synskadade, Synskadade i Malmöhus län, Anna och Edvin Berger's Foundation, and the Lars Hiertas Minne Foundation.
Footnotes
 Disclosure: H. Morén, None; P. Undrén, None; B. Gesslein, None; G.K. Olivecrona, None; S. Andreasson, None; M. Malmsjö, None
Footnotes
 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The authors thank Christer Eriksson and ev3 for valuable support and Matthias Götberg at the Department of Cardiology in Lund, Sweden, for excellent input into this study. 
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Figure 1.
 
Angiogram of the left common carotid artery in a pig. (A) Basal view. (B) Lateral view. (C) Enlargement of the inset in (B). The (1) ascending pharyngeal artery originates, as a small side branch, from the (2) common carotid artery and feeds the (3) rete mirabilis, which then converges to form the intracranial carotid artery. The (4) external carotid artery is a continuity of the common carotid artery. The (5) maxillary artery branches from the external carotid artery. The maxillary artery gives off the (6) lingual, the (7) auricular, the (8) facial, and the (9) buccinator arteries. The maxillary artery gives rise to the (10) infraorbital artery. The (11) ophthalmic artery branches off the infraorbital artery. After having accessed the ophthalmic artery, injection of contrast will produce a characteristic half-moon–shaped outline of the retina (arrows). The ophthalmic artery gives off the main ciliary artery, (12) from which the retinal artery branches.
Figure 1.
 
Angiogram of the left common carotid artery in a pig. (A) Basal view. (B) Lateral view. (C) Enlargement of the inset in (B). The (1) ascending pharyngeal artery originates, as a small side branch, from the (2) common carotid artery and feeds the (3) rete mirabilis, which then converges to form the intracranial carotid artery. The (4) external carotid artery is a continuity of the common carotid artery. The (5) maxillary artery branches from the external carotid artery. The maxillary artery gives off the (6) lingual, the (7) auricular, the (8) facial, and the (9) buccinator arteries. The maxillary artery gives rise to the (10) infraorbital artery. The (11) ophthalmic artery branches off the infraorbital artery. After having accessed the ophthalmic artery, injection of contrast will produce a characteristic half-moon–shaped outline of the retina (arrows). The ophthalmic artery gives off the main ciliary artery, (12) from which the retinal artery branches.
Figure 2.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). (B) Positioning of a balloon-catheter (arrow) in the ophthalmic artery (asterisk), with its tip at the branching of the main ciliary artery. (C) Angiogram during balloon occlusion of the ophthalmic artery. Note that contrast does not enter the ophthalmic artery during balloon occlusion.
Figure 2.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). (B) Positioning of a balloon-catheter (arrow) in the ophthalmic artery (asterisk), with its tip at the branching of the main ciliary artery. (C) Angiogram during balloon occlusion of the ophthalmic artery. Note that contrast does not enter the ophthalmic artery during balloon occlusion.
Figure 3.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). Both the ophthalmic artery (asterisk) and the main ciliary artery, with the branching of the retinal artery (arrow), are visible. Right: enlargement of the inset in the left panel, showing the main ciliary artery and the branching of the retinal artery (arrow). (B) Positioning of the catheter for injection of liquid embolic agent (arrow) in the main ciliary artery. Asterisk: ophthalmic artery. (C) Result after injection of the radiopaque liquid embolic agent in the main ciliary artery and the retinal artery (arrow). (D) Angiogram during liquid embolic agent occlusion of the main ciliary and retinal artery. Note that contrast does not enter the ophthalmic or main ciliary artery.
Figure 3.
 
(A) Angiogram of the left common carotid artery in a pig (lateral projection). Both the ophthalmic artery (asterisk) and the main ciliary artery, with the branching of the retinal artery (arrow), are visible. Right: enlargement of the inset in the left panel, showing the main ciliary artery and the branching of the retinal artery (arrow). (B) Positioning of the catheter for injection of liquid embolic agent (arrow) in the main ciliary artery. Asterisk: ophthalmic artery. (C) Result after injection of the radiopaque liquid embolic agent in the main ciliary artery and the retinal artery (arrow). (D) Angiogram during liquid embolic agent occlusion of the main ciliary and retinal artery. Note that contrast does not enter the ophthalmic or main ciliary artery.
Figure 4.
 
Representative examples of the mfERG recordings. Left: topographical maps in which the optic nerve head (large arrows) and visual streak (small arrows) can be visualized. Right: individual recordings. mfERG recordings were obtained (A) before catheterization, (B) during balloon-occlusion of the ophthalmic artery, over the branching of the main ciliary artery, and (C) after injection of liquid embolic agent into the main ciliary and retinal arteries. Note that occlusion of the ophthalmic artery causes incomplete retinal ischemia with decreased amplitude and increased implicit time. Occlusion of the ciliary and retinal artery causes complete retinal ischemia with flattening of the mfERG traces.
Figure 4.
 
Representative examples of the mfERG recordings. Left: topographical maps in which the optic nerve head (large arrows) and visual streak (small arrows) can be visualized. Right: individual recordings. mfERG recordings were obtained (A) before catheterization, (B) during balloon-occlusion of the ophthalmic artery, over the branching of the main ciliary artery, and (C) after injection of liquid embolic agent into the main ciliary and retinal arteries. Note that occlusion of the ophthalmic artery causes incomplete retinal ischemia with decreased amplitude and increased implicit time. Occlusion of the ciliary and retinal artery causes complete retinal ischemia with flattening of the mfERG traces.
Figure 5.
 
Photographs from a porcine eye in which liquid embolic agent was injected extensively in the ciliary and retinal arteries via transfemoral catheterization. (A) Photograph of the posterior portion of the dissected eye. The ciliary arteries are filled with the black liquid embolic agent. (B) Photograph of the eyecup with the retina, optic nerve head, and arteries. The retinal arteries are pale with no glue and are sparse with blood segmented in the vessels. (C) Angiogram of the same eye showing the ophthalmic artery (asterisk) and the ciliary and retinal arteries, all which are filled with the radiopaque liquid embolic agent (arrow).
Figure 5.
 
Photographs from a porcine eye in which liquid embolic agent was injected extensively in the ciliary and retinal arteries via transfemoral catheterization. (A) Photograph of the posterior portion of the dissected eye. The ciliary arteries are filled with the black liquid embolic agent. (B) Photograph of the eyecup with the retina, optic nerve head, and arteries. The retinal arteries are pale with no glue and are sparse with blood segmented in the vessels. (C) Angiogram of the same eye showing the ophthalmic artery (asterisk) and the ciliary and retinal arteries, all which are filled with the radiopaque liquid embolic agent (arrow).
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