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. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: Exp Eye Res. 2015 Oct 28;143:141–147. doi: 10.1016/j.exer.2015.09.018

The Effect of Intravitreal Vascular Endothelial Growth Factor on Inner Retinal Oxygen Delivery and Metabolism in Rats

Norman P Blair a,✉, Justin Wanek a, Pang-yu Teng a,1, Mahnaz Shahidi a
PMCID: PMC4699657  NIHMSID: NIHMS736112  PMID: 26518179

1.0 Introduction

Vascular endothelial growth factor (VEGF) is a signal protein that has potent mitogenic and survival activity for endothelial cells and powerful vascular permeability-enhancing properties. (Ferrara, 2004; Nishijima et al., 2007) Thus, it plays an important role in embryonic retinal vascular development, pathologic retinal vascular leakage, and neovascularization of the choroid, optic nerve, retina, and iris. (Ferrara, 2004) Accordingly, inhibitors of VEGF are now the treatments of choice for many cases of macular edema, (Brown et al., 2011; Campochiaro et al., 2011; Diabetic Retinopathy Clinical Research et al., 2010; Nguyen et al., 2010) choroidal neovascularization (Gragoudas et al., 2004; Rosenfeld et al., 2006) and neovascularization of the retina, optic nerve and iris. (Ernst et al., 2012; Grisanti et al., 2006; Martinez-Zapata et al., 2014)

Retinal hypoxia/ischemia, via hypoxia inducible factor (HIF), (Ozaki et al., 1999) is a major stimulator of VEGF. (Aiello et al., 1995; Miller et al., 1994; Pe'er et al., 1995) This suggests that VEGF may play a role in counteracting hypoxia and the injurious effects that supervene if the hypoxia is severe enough. There are several ways by which VEGF could mitigate hypoxic effects. First, by stimulating endothelial proliferation and vessel growth, VEGF could lead to better perfusion and alleviate retinal hypoxia. However, neovascularization often fails to vascularize ischemic tissue and instead can lead to deleterious effects such as hemorrhage and fibrosis. Furthermore, elevated VEGF over time can stimulate excessive endothelial proliferation that can occlude capillaries. (Hofman et al., 2001; Tolentino et al., 2002) Second, VEGF has been shown to have a survival role on retinal neural cells. (Saint-Geniez et al., 2008) Third, VEGF could increase retinal blood flow and oxygen delivery to the inner retina (DO2). VEGF has been shown to cause retinal vasodilation, vascular tortuosity and increased blood flow in animals. (Ali Rahman et al., 2011; Ameri et al., 2007; Arana et al., 2012; Clermont et al., 1997; Ozaki et al., 1997) Several studies are available on retinal vascular changes after treatment of patients with VEGF inhibitors. A reduction in vessel diameters (Fontaine et al., 2011; Papadopoulou et al., 2009) and reductions in retrobulbar flow velocities have been observed. (Bonnin et al., 2010; Hosseini et al., 2012; Mete et al., 2010; Toklu et al., 2011) Nitta and coworkers found that the retinal blood flow measured by laser speckle flowgraphy decreased in patients treated for diabetic macular edema with an anti-VEGF agent. (Nitta et al., 2014) These observations imply that prior to treatment VEGF had increased retinal blood flow and DO2. On the other hand, Fontaine and coworkers found no change in retinal blood flow by laser Doppler flowmetry after anti-VEGF therapy for age-related macular degeneration even though the retinal arterioles became narrower. (Fontaine et al., 2011) Barak and coworkers found increased blood velocity with the retinal function imager after anti-VEGF treatment, and they attributed this to suppression of vasodilation. (Barak et al., 2012) We have not found in the literature any measurements of DO2 after the introduction of VEGF into the vitreous. Fourth, VEGF could down-regulate inner retinal oxygen metabolism (MO2), as HIF does, (Kim et al., 2006) so that the tissue can adapt to mild reductions in oxygen availability. Reduced MO2 might be expected to contribute to ischemic preconditioning and the suppression of cell death pathways, processes in which VEGF is thought to play a role. (Evans et al., 2008; Jin et al., 2001; Parcellier et al., 2003; Wick et al., 2002) However, we have not found any measurements in the literature on the effects of VEGF on inner retinal MO2.

We hypothesized that VEGF increases DO2, which could be advantageous in conditions characterized by hypoxia, but that it does not alter MO2. We tested these hypotheses using the methods we have developed to measure DO2 and MO2 in rats. (Wanek et al., 2011; Wanek et al., 2013, 2014)

2.0 Methods

2.1 Animals

Ten Long Evans pigmented rats (weight: 483 ± 127 g, mean ± SD) were used in the study. The rats were treated in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (5 mg/kg). Following the method of Miyamoto and co-workers, the right eye of each rat received an intravitreal injection of VEGF A (R & D Systems, Minneapolis, MN) while the fellow eye received vehicle (BSS) alone. (Miyamoto et al., 2000) The dose was increased from the concentration employed by Miyamoto et al (50 ng in 5 µl) to 250 ng in 5 µl of BSS because of the extremely rapid clearance of VEGF from the vitreous as recently reported. (Lee et al., 2010) Data acquisition was performed 24 hours after VEGF injection. The rats were reanesthetized with intraperitoneal injections of ketamine and xylazine. Additional injections of ketamine (20 mg/kg) and xylazine (1 mg/kg) were given to maintain anesthesia as required. Rats were ventilated mechanically with room air with the use of an endotracheal tube connected to a small animal ventilator (Harvard Apparatus, Inc., South Natick, MA). To monitor the animal's physiological condition, the femoral artery was cannulated and a catheter was attached to draw blood and connect a pressure transducer. Systemic arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2), and pH were measured with a blood gas analyzer (Radiometer, Westlake, OH) 5 to 10 minutes after initiation of ventilation. Ventilation parameters, including the respiratory rate and minute volume, were adjusted until the PaCO2 was within the normocapnic range. (West, 2007) Hemoglobin concentration (HgB) was also measured with a hematology system (Siemens, Tarrytown, NY) from arterial blood. Blood pressure (BP) and heart rate (HR) were monitored continuously with a data acquisition system (Biopac Systems, Goleta, CA) linked to the pressure transducer.

Before imaging, the rat was placed in an animal holder with a copper tubing water heater, which maintained the body temperature at 37°C. The pupils were dilated with 2.5% phenylephrine and 1% tropicamide. A glass cover slip with 1% hydroxypropyl methylcellulose was applied to the cornea to eliminate its refractive power and prevent dehydration. For retinal vascular PO2 imaging, an oxygen-sensitive molecular probe, Pd-porphine (Frontier Scientific, Logan, UT), was dissolved (12 mg/mL) in bovine serum albumin solution (60 mg/mL) and administered through the femoral arterial catheter (20 mg/kg). For retinal blood velocity imaging, 2-µm polystyrene fluorescent microspheres (Invitrogen, Grand Island, NY) were injected through the catheter. Typically, two to three injections of the microspheres were given, and each injection was approximately 0.4 mL (105 microspheres/mL).

2.2 Retinal Hemodynamics Imaging

Our previously described prototype blood flow imaging system (Wanek et al., 2011) was used for red-free and fluorescent microsphere imaging to assess venous blood vessel diameter and velocity, respectively. A slit lamp biomicroscope with standard light illumination (Carl Zeiss, Oberkochen, Germany) was equipped with a green filter (540 ± 5 nm; Edmund Optics, Barrington, NJ) for red-free retinal imaging, and a 488-nm diode laser (Melles Griot, Carlsbad, CA), coupled with an emission filter (560 ± 60 nm; Spectrotech, Inc., Saugus, MA) for fluorescent microsphere imaging. Images were captured with a high-speed electron multiplier charge coupled device camera (QImaging, Surrey, Canada). Red-free retinal images were obtained using the full resolution of the camera (1002 × 1004 pixels). For fluorescent images, the camera sensor was binned to maximize the frame rate to 108 Hz, allowing the motion of the microspheres to be resolved in time, but with lower spatial resolution (248 × 250 pixels). Multiple image sequences, each 5 seconds in duration, were recorded over several minutes.

Diameters of all individual retinal arteries (DAind) and veins (DVind) were measured from red-free images over a fixed vessel length (200 µm) that extended between approximately 300 and 500 µm from the center of the optic disk. DAind and DVind were obtained based on the average full width at half maximum of 12 intensity profiles perpendicular to the blood vessel axis. In control eyes 5.2 ± 1.0 arteries and 5.2 ± 1.1 veins were measured, whereas 5.5 ± 1.1 arteries and 5.4 ± 1.0 veins were included from VEGF-injected eyes. For each rat, a mean arterial (DA) and venous diameter (DV) was calculated from all DAind and all DVind values, respectively. Blood velocity in all individual veins (Vind) was measured by manually tracking displacements of the microspheres over time, following our previously reported methodology. (Wanek et al., 2011) Typically, three to five image sequences were analyzed to derive Vind, which was determined by averaging 20 – 30 microsphere velocity measurements in each vein. The number of measurements used to obtain each Vind was contingent on the number of microspheres that could be visualized in the image sequences. In each rat, a mean velocity (V) was derived from all Vind measurements. V was measured in veins because they are less affected by pulsation and have larger diameters as compared with arteries. Blood flow in each major vein was calculated from DVind and Vind measurements (Vind*π*DVind2/4) and summed over all veins to provide a measure of the total venous blood flow in the retinal circulation (F) in that animal. Since the retinal circulation is considered to be an end-artery system, (Levin and Adler, 2011) the venous blood flow was taken to be equal to the total arterial or total retinal blood flow. Measurements of F were obtained approximately 15 minutes following PO2 imaging, while the physiological state of the animals was relatively stable, as indicated by the continuous monitoring of BP, HR, and the use of constant ventilation parameters.

2.3 Retinal Vascular PO2 Imaging

Retinal vascular PO2 was measured using our established optical section phosphorescence lifetime imaging system. (Shahidi et al., 2006; Shahidi et al., 2009) A laser line was projected on the retina after intravenous injection of the Pd-porphine probe and an optical section phosphorescence image was acquired with an intensified charge-coupled device camera. Due to the angle between the excitation laser beam and imaging path, phosphorescence emissions from the retinal vessels were depth-resolved from the underlying choroid. Phosphorescence lifetimes in the retinal vessels were determined using a frequency-domain approach and converted to PO2 measurements using the Stern-Volmer equation. (Lakowicz et al., 1992; Shonat and Kight, 2003) PO2 was measured in all major retinal arteries (PO2Aind) and retinal veins (PO2Vind) at locations within three optic disc diameters (~600 µm) from the edge of the optic nerve head. Three repeated PO2Aind and PO2Vind measurements were averaged per blood vessel. The mean and SD of the PO2Aind values for arteries (PO2A) and PO2Vind values for veins (PO2V) were calculated for each animal.

The oxygen content of blood (O2ind) was calculated for each individual major retinal artery and vein as the sum of oxygen bound to hemoglobin and dissolved in blood: (Pittman, 2011) O2ind = SO2*C*HgB + PO2*k, where SO2 is the oxygen saturation (%), C is the oxygen-carrying capacity of hemoglobin (1.39 mL O2/g), (Nathan and Singer, 1999) and k is the oxygen solubility in blood (0.003 mL O2/dL·mm Hg. (Pittman, 2011) SO2 was calculated from the hemoglobin dissociation curve in rat (Cartheuser, 1993) by using the measured blood PO2 and pH values. Last, in each animal mean arterial (O2A) and venous (O2V) oxygen contents were determined from O2ind measurements. The arteriovenous oxygen content difference (O2A-V) was calculated as: O2A-V= O2A−O2V.

2.4 Oxygen Delivery and Oxygen Metabolism

DO2 was determined from the F and O2A measurements according to the following equation: (Pittman, 2011) DO2 = F* O2A. MO2 was calculated from measurements of F and O2A-V, using Fick's principle: (Pittman, 2011) MO2 = F * O2A-V.

2.5 Statistical Analysis

The data were analyzed with the paired Student’s t test. Statistical significance was accepted at P less than 0.05.

3.0 Results

3.1 Systemic Physiologic Factors

BP and HR were 105 ±17 mm Hg and 235 ± 44 beats/min, respectively. The systemic PaO2, PaCO2 and pH were 86 ± 7 mm Hg, 40 ± 9 mm Hg and 7.42 ± 0.06, respectively. These indicate that the rats had a normal systemic physiologic condition during the experiments.

3.2 Retinal Hemodynamic Factors

The results of the retinal hemodynamic factors are presented in Table 1. DA in the control and VEGF-injected eyes were 42 ± 6 and 44 ± 5 µm, respectively (P=0.03), an increase of 4%. A greater effect was observed in DV. The values in the control and eyes receiving VEGF were 50 ± 6 and 61 ± 12 µm, respectively (P=0.008), an increase of 22%. Figure 1 depicts red-free fundus images of normal and VEGF-injected eyes. Vasodilation is visible in the VEGF-injected eyes, especially in the veins. No significant difference was found in V, which were 11.7 ± 2.4 and 10.9 ± 2.6 mm/s in the control and VEGF-injected eyes, respectively (P=0.21). The largest effect of VEGF was seen in F. These were 7.3 ± 1.6 and 10.6 ± 3.9 µl/min without and with VEGF injection, respectively, (P=0.007), an increase of 45%.

Table 1.

Retinal Vascular Factors in Rat Eyes with Intravitreally Injected Vascular Endothelial Growth Factor and in their Control Fellow Eyes

Control VEGF P Value
DA (µm) 42 ± 6 44 ± 5 0.03
DV (µm) 50 ± 6 61 ± 12 0.008
V (mm/s) 11.7 ± 2.4 10.9 ± 2.6 0.21
F (µl/min) 7.3 ± 1.6 10.6 ± 3.9 0.007
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VEGF = Vascular Endothelial Growth Factor, DA = Retinal Arterial Diameter, DV = Retinal Venous Diameter, V = Venous Blood Velocity, F = Total Inner Retinal Blood Flow

Figure 1.

Figure 1

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Red-free images of the two peripapillary areas of a rat, one without (left) and one with (right) intravitreally injected vascular endothelial growth factor (VEGF). Note the increase in vessel diameters, especially in the veins, induced by VEGF.

3.3 Retinal Vascular Oxygenation Factors

The results of the vascular oxygen factors are displayed in Table 2. A statistically significant difference was found between control and VEGF-injected eyes only in O2A-V. PO2A were 46 ± 12 and 45 ± 9 mm Hg in the control and VEGF-injected eyes, respectively (P=0.41). PO2V were 28 ± 6 in the control eyes and 31 ± 6 mm Hg in the eyes receiving VEGF (P=0.11). In the control and VEGF-injected eyes O2A were 12.9 ± 2.8 and 12.8 ± 2.4 mL O2/dl, respectively (P=0.92). O2V were 6.9 ±2.5 and 8.1 ± 2.3 mL O2/dl in the control and eyes administered VEGF, respectively (P=0.12). O2A-V were 6.0 ± 1.7 and 4.8 ± 1.1 mL O2/dl in the control and VEGF-injected eyes, respectively (P=0.05).

Table 2.

Retinal Vascular Oxygen Factors in Rat Eyes with Intravitreally Injected Vascular Endothelial Growth Factor and in their Control Fellow Eyes

Control VEGF P Value
PO2A (mm Hg) 46 ± 12 45 ± 9 0.41
PO2V (mm Hg) 28 ± 6 31 ± 6 0.11
O2A (mL O2/dl) 12.9 ± 2.8 12.8 ± 2.4 0.92
O2V (mL O2/dl) 6.9 ± 2.5 8.1 ± 2.3 0.12
O2A-V (mL O2/dl) 6.0 ± 1.7 4.8 ± 1.1 0.05
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VEGF = Vascular Endothelial Growth Factor, PO2A = Retinal Arterial PO2, PO2V = Retinal Venous PO2, O2A = Retinal Arterial Oxygen Content, O2V = Retinal Venous Oxygen Content, O2A-V = Retinal Arteriovenous Content Difference

3.4 Oxygen Delivery and Oxygen Metabolism

The results of oxygen delivery and oxygen metabolism are presented in Table 3 and depicted graphically in Figure 2. This figure shows that in 9 of 10 rats DO2 was greater in the VEGF-injected eye than in the control eye. DO2 were 950 ± 340 and 1380 ± 650 nL O2/min in the control and eyes receiving VEGF, respectively (P=0.005), an increase of 45 %. In contrast, no difference was observed in MO2 between the two groups. MO2 were 440 ± 150 and 490 ± 190 nL O2/min in the control and VEGF-injected eyes, respectively (P=0.31).

Table 3.

Inner Retinal Oxygen Delivery and Metabolism in Rat Eyes with Intravitreally Injected Vascular Endothelial Growth Factor and in their Control Fellow Eyes

Control VEGF P Value
DO2 (nL O2/min) 950 ± 340 1380 ± 650 0.005
MO2 (nL O2/min) 440 ± 150 490 ± 190 0.31
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VEGF = Vascular Endothelial Growth Factor, DO2 = inner retinal oxygen delivery, MO2 = inner retinal oxygen metabolism

Figure 2.

Figure 2

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Inner Retinal Oxygen Delivery (DO2) and Oxygen Metabolism (MO2) in Rat Eyes with Intravitreally Injected Vascular Endothelial Growth Factor (VEGF) and in their Control Fellow Eyes. Lines join values from the two eyes of each animal.

4.0 Discussion

In the present study VEGF induced an increase in DO2 while MO2 remained unchanged. The increase in DO2 was mainly caused by an increase in F, since we found no change in O2A. The increase in F, in turn, was caused by an increase in DV, since no change in V was observed. Because VEGF stimulates nitric oxide, (Fukumura et al., 2001) it is not surprising that both the arteries and veins were dilated, as many have found in animals injected with VEGF intravitreally. As expected O2A was unchanged by VEGF because it is dominated by systemic phenomena acting before blood enters the retina, whereas O2V is dominated by the extraction of oxygen during the transit of the blood from artery to vein, namely MO2. When DO2 increases while MO2 remains unchanged, the amount of oxygen left in the veins to exit the eye must increase, accounting for the smaller O2A-V we observed in the eyes injected with VEGF.

No measurements of DO2 in the retina have been reported until recently because methods have not been available to measure both F and O2A in the same experiment. However, significant information on DO2 can be inferred from F measurements because O2A tends to be constant in the absence of systemic changes, such as anemia or low FiO2. Clermont and coworkers reported increases in retinal blood flow in rats after intravitreal administration of VEGF. (Clermont et al., 1997) They measured the vessel diameters and the mean circulation time of injected fluorescein to obtain their results in units of pixel2 per time. Inner retinal blood flow equals the volume of the vascular tree divided by the mean circulation time. (Eberli et al., 1979) Thus, this method gives results that are related to actual blood flow only to the extent that the square of the vessel diameters represents the vascular volume. Several studies have looked at blood flow in patients who had been treated with inhibitors of VEGF. However, the level of VEGF at baseline and the completeness of inhibition at the time of measurement were unknown, so only semi-quantitative conclusions can be drawn about the effect of VEGF on retinal blood flow. None have combined blood flow estimates with oxygen measurements.

Studies on the metabolic effects of VEGF have not suggested a major influence on energy generation. (Evans et al., 2008; Jin et al., 2001; Parcellier et al., 2003; Wick et al., 2002) As we hypothesized, we found no alterations in inner retinal MO2, even though DO2 increased. No previous measurements are available on the effect of VEGF on inner retinal MO2. Again, this is related to limitations in methods to acquire this information.

Much important information has been obtained on oxygen in the retina with previous methods. Oxygen consumption can be measured in the outer retina with oxygen sensitive microelectrodes and slit-beam phosphorescent imaging in animals assuming one dimensional oxygen diffusion from the choroid. (Linsenmeier, 1986; Linsenmeier and Braun, 1992; Pournaras, 1995; Wanek et al., 2012; Yu and Cringle, 2001) However, the validity of this assumption is questionable in the inner retina except under certain experimental conditions. (Alder et al., 1990; Braun et al., 1995) Methods to assess retinal oxygenation including MRI and oximetry have yielded great contributions to our understanding of retinal oxygenation, (Choudhary et al., 2013; Hammer et al., 2009; Hardarson, 2013; Kashani et al., 2014; Klefter et al., 2014; Man et al., 2013; Shahidi et al., 2013; Trick and Berkowitz, 2005; Yi et al., 2013; Zhang et al., 2011) but these do not quantify MO2. Recently, Palkovits and coworkers have published results obtained by the combined use of retinal vascular oximetry, vessel caliber analysis and blood velocity measurements using laser Doppler velocimetry in healthy subjects. (Palkovits et al., 2014a; Palkovits et al., 2014b) We anticipate that much useful information will be obtained with these methods in the near future.

Our results on the effects of VEGF on DO2 and MO2 may be relevant to sight threatening diabetic retinopathy in which the presence of nonperfusion, hypoxia and elevated VEGF is indisputable. Their significance in the early stages of human diabetic retinopathy and retinopathy in rats with diabetes is less clear. VEGF levels have been found to be elevated in diabetic rats within three months of diabetes. (Gong et al., 2013; Kusari et al., 2010) However, we have reported no changes in DO2 or MO2 in rats after 6 weeks of diabetes and no intraretinal hypoxia was found in diabetic rats, at least up to 12 weeks of diabetes. (Lau and Linsenmeier, 2014; Wright et al., 2011). Presumably, in this case VEGF is stimulated by factors other than hypoxia and at levels low enough not to enhance DO2.

One limitation of the current study was lack of knowledge about the amount of VEGF in the retina 24 hours after intravitreal injection. This time interval allowed diffusion of VEGF from the vitreous into the retina. The amount of VEGF in the retina depends on the initial vitreal concentration, half-life, and time. The amount of VEGF we injected was 250 ng, which is within the range of the amounts (2–10,000 ng) employed by other investigators. (Ameri et al., 2007; Arana et al., 2012; Clermont et al., 1997; Hofman et al., 2001; Lee et al., 2010; Miyamoto et al., 2000; Tolentino et al., 1996) Loss of VEGF from the vitreous is extremely rapid. The half-life of VEGF in the rabbit vitreous has been reported to be 2.46 hours.(Lee et al., 2010) If VEGF in rats has the same half-life, approximately nine half-lifes would have occurred by the time of our measurements at 24 hours after injection. In this case, the VEGF concentration would be reduced to about 1/500 of the initial value, which would be about 10 ng/ml assuming a rat vitreous volume of 50 µl. This is well within the range of maximal concentrations reported in diseased human eyes (3 – 5000 ng/ml). (Adamis et al., 1994; Aiello et al., 1994; Brooks et al., 2004; Funatsu et al., 2002; Malecaze et al., 1994; Malik et al., 2005; Ogata et al., 2002; Simo et al., 2002; Watanabe et al., 2005) While the half-life of intravitreal VEGF in rats may not be identical to that in rabbits, there still would have been a dramatic reduction of the initial intravitreal concentration of VEGF by the time of our measurements. Further studies are needed to determine the optimal dose and time interval for evaluating VEGF-induced changes in retinal oxygen delivery and oxygen metabolism.

We have formed hypotheses based on the notion that VEGF has effects that counter those of hypoxia. This seems reasonable since VEGF is stimulated by HIF and it induces nitric oxide. Nonetheless, intravitreal administration of VEGF causes a retinopathy resembling ischemic retinopathies characterized by vascular tortuosity, edema, hemorrhages, microaneurysms, capillary occlusion and neovascularization. (Ali Rahman et al., 2011; Ameri et al., 2007; Arana et al., 2012; Hofman et al., 2001; Ozaki et al., 1997; Tolentino et al., 2002; Tolentino et al., 1996) Neovascularization and edema are not surprising based on the known mitogenic and permeability-enhancing actions of VEGF. It appears that with high levels and time VEGF may actually exacerbate retinal hypoxia, at least locally, by inducing excessive endothelial proliferation that can occlude capillaries and by inducing retinal edema that will lengthen the diffusion distance between the capillaries and cells. Thus, there is complexity to the actions of VEGF in that it can both counteract and exacerbate retinal hypoxia.

Highlights.

  • Vascular endothelial growth factor (VEGF) was injected intravitreally in rats.

  • VEGF induced vasodilation in retinal arteries and veins.

  • Blood velocity and vascular oxygen content were not altered by VEGF.

  • VEGF increased retinal blood flow and oxygen delivery.

  • Retinal oxygen metabolism was not altered by VEGF.

Acknowledgements

This study was supported by the National Eye Institute, Bethesda, MD, EY017918 (MS) and EY001792 (UIC), Research to Prevent Blindness, New York, NY, senior scientific investigator award (MS) and an unrestricted departmental award. We acknowledge the technical support of Marek Mori and Tara Nguyen.

Footnotes

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Contributor Information

Norman P. Blair, Email: npblair@uic.edu.

Justin Wanek, Email: wanek_justin@yahoo.com.

Pang-yu Teng, Email: pangyuteng@gmail.com.

Mahnaz Shahidi, Email: mahnshah@uic.edu.

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