Defining the minimally effective dose and schedule for parenteral hydrogen sulfide: long-term benefits in a rat model of hindlimb ischemia
© Langston and Toombs; licensee BioMed Central. 2015
Received: 5 February 2015
Accepted: 6 April 2015
Published: 16 April 2015
Peripheral arterial disease (PAD) affects millions of Americans and leads to critical limb ischemia (CLI) in the most severe cases. Investigators have demonstrated the utility of hydrogen sulfide for restoring perfusion in rodent models of chronic ischemia. We sought to determine the minimum effective dose (MED) of sulfide necessary to restore perfusion in the rat hindlimb, to assess the persistence of limb perfusion after cessation of treatment, and to compare perfusion measurements between laser doppler and ultrasound methods.
In 3 separate experiments, sodium sulfide (1.0, 0.5, or 0.25 mg/kg twice daily for 14 days, 0.25 mg/kg twice daily for 7 days, 0.5 mg/kg once daily for 7 days, or 0.25 mg/kg twice daily for 3 days) or vehicle was administered after left femoral artery ligation and transection. Hindlimb perfusion was assessed by laser doppler flowmetry and contrast enhanced ultrasound over the duration of each study, and cellular proliferation and vascular density were assessed by immunohistochemical means in the initial experiment.
Intravenous sodium sulfide at 0.25, 0.5, or 1.0 mg/kg twice daily for 2 weeks significantly enhanced the recovery of blood flow to the ischemic hindlimb by 7 days. The enhancement of blood flow with 1.0 mg/kg dosing was coincident with an increase in cellular proliferation and vascular density in the ischemic tissue. In a final experiment, i.v. administration of sodium sulfide at 0.5 mg/kg once daily for 7 days or 0.25 mg/kg twice daily for 7 days significantly elevated blood flow and skeletal muscle perfusion in the ischemic hindlimb, whereas 0.25 mg/kg twice daily for 3 days had no effect. This enhancement of blood flow appeared long lived, as blood flow remained elevated 3 weeks after cessation of treatment.
These data, together with other published observations, demonstrate the efficacy of hydrogen sulfide in restoring perfusion to chronically ischemic tissue and establish a minimum efficacious dose in the rat hindlimb model.
Peripheral arterial disease (PAD) is characterized by a narrowing of the arteries in peripheral vessels caused by atherosclerotic plaque formation, resulting in decreased blood flow to distal appendages. This disorder affects 8 to 12 million Americans and represents a significant financial health care burden that will rise concomitantly with the rates of obesity and diabetes . Critical limb ischemia (CLI), characterized by pain at rest, ulcerations, and/or gangrene in the extremity, is considered an end stage of PAD, with the majority of these patients undergoing revascularization (bypass or angioplasty) or amputation as long as they can tolerate surgery [2,3]. Currently, there is no established nonsurgical treatment regimen for CLI patients.
Hydrogen sulfide (H2S) plays a number of important roles in cardiovascular physiology as a regulator of inflammation, angiogenesis, vessel tone, and redox potential [4-6]. It has also been shown to be protective in animal models of cardiovascular disease including heart failure [7-9], hypertension [10,11], atherosclerosis [12,13], and ischemia reperfusion injury in multiple organs and tissues [14,15]. Mechanistically, sulfide is thought to mediate these effects in a variety of ways. As a second messenger and signaling molecule, sulfide is thought to modulate cellular function via reversible cysteine s-sulfhydration of a number of enzymes, transcription factors, and cytoskeletal elements [16-18]. Another purported cytoprotective mechanism stems from observations that sulfide at lower concentrations can reversibly reduce the oxygen affinity of cytochrome c oxidase, effectively dampening cellular respiration [19,20]. The nucleophilic nature of H2S is also thought to contribute to its observed efficacy via the attenuation of oxidative stress and reactive oxygen species (ROS) signaling .
Therapeutic angiogenesis refers to the pharmacologic induction of neovascularization to restore blood flow and oxygenation in ischemic tissue, and published observations suggest hydrogen sulfide could be such a pharmacophore. H2S stimulates endothelial cell migration, proliferation, and tube formation in vitro, and has been shown to stimulate vascular growth in the chicken chorioallantoic membrane and into matrigel plugs implanted in vivo in mice [5,22]. Furthermore, hydrogen sulfide has been shown to stimulate vascular growth in ischemic heart tissue and improve cardiac function in rodent models of heart failure [8,23]. The molecular mechanisms underlying these effects are being investigated, and evidence suggests that H2S enhances nitric oxide production and bioavailability [24,25], increases HIF-1α mediated gene expression (including VEGF and its receptors) [24,26], and can activate VEGFR2 directly via reduction of a vicinal disulfide bond .
To date, several investigators have demonstrated the capacity of hydrogen sulfide to revascularize and restore blood flow to skeletal muscle in rodent models of hindlimb ischemia (HLI). Wang and colleagues used fluorescent microspheres to assess the effect of NaHS on rat hindlimb perfusion, and Bir et al. used laser doppler flowmetry to demonstrate the beneficial effect of Na2S on perfusion of the mouse hindlimb [24,28]. Both studies provided evidence suggesting that H2S mediates these proangiogenic effects via the upregulation of VEGF signaling. In the current study, we sought to determine a minimum dose and schedule of parenteral hydrogen sulfide sufficient to restore perfusion in the rat hindlimb. Utilizing contrast enhanced ultrasound to corroborate laser doppler flowmetry results, we demonstrate here that a twice daily intravenous infusion of 0.25 mg/kg sodium sulfide over as few as 7 days was sufficient to significantly enhance perfusion in the ischemic hindlimb. Moreover, the effect on perfusion persisted several weeks after cessation of treatment.
Male CD rats, 350–450 g with jugular vein catheters (JVC), were purchased from Charles River (Wilmington, MA). All experiments were approved by the internal Ethical Committee for Animal Care and Use. All animals were singly housed and allowed to acclimate in house for at least 48 hours prior to study entry. The facility was kept at a temperature of 21°C, humidity of 45-55%, under a 12 hour light/dark cycle. Animals had access to food and water ad libitum.
Under 1.5% isoflurane, animal fur was removed from the distal hind limbs using depilatory cream, and blood flow was measured as described below. An incision was made in the left groin, and the femoral artery was carefully dissected away from the femoral vein, femoral nerve, and surrounding fascia. The femoral artery was ligated at two points with silk suture just distal to the inguinal ligament, and the vessel was then transected between the two ligation points. The incision in the groin was stapled closed, and hind limb blood flow was measured again as described below.
Laser doppler flowmetry
A laser doppler flow meter (Product# ML191, ADInstruments, Colorado Springs, CO) was used in conjunction with either a miniature surface flow probe (Product# MSP300XP, ADInstruments, Colorado Springs, CO) or a fine needle flow probe (Product# MNP110XP, ADInstruments, Colorado Springs, CO) to measure hind limb blood flow. Blood flow was recorded and analyzed using a PowerLab 8/30 (Product# ML870/P, Colorado Springs, CO) with LabChart software, and a heat lamp was used to maintain core body temperature while all blood flow measurements were obtained. While under 1.5 % isoflurane, flow measurements were taken from the proximal, medial, and distal portions of each calf twice, for a total of 6 measurements per calf that were averaged to give a single value. Blood flow in the ischemic (left) limb was normalized to blood flow in the nonischemic (right) limb.
Contrast enhanced ultrasound measurements of hindlimb perfusion
A Vevo 770 high-resolution ultrasound imaging system (VisualSonics, Toronto, Ontario, Canada) equipped with a 15–45 MHz RMV scanhead (Model# RMV707B, VisualSonics, Toronto, Ontario, Canada) was used to visualize the gastrocnemius and measure perfusion within it. While under 1.5% isoflurane, a lateral section of each gastrocnemius parallel to the fibula was visualized. Once a satisfactory image was obtained, 150 μL of a contrast microbubble solution containing 3.0 x 108 microbubbles were given as a bolus via the jugular vein catheter, and the change in contrast visualized within each gastrocnemius muscle was used to indicate perfusion in the ischemic hindlimb relative to the nonischemic limb. A heat lamp and heated stage were used to maintain core temperature of each animal over the course of measurements.
Immunohistochemical assessment of vascular density and cellular proliferation
Upon completion of the experimental protocol, each animal was euthanized by exsanguination under 3.0% isoflurane anesthesia. An incision was then made in the left ventricle, and 10% buffered neutral formalin was pushed through the lower torso via the abdominal aorta just proximal to the iliac bifurcation. The left (ischemic) and right (nonischemic) gastrocnemius muscles were then excised and placed in 10% buffered neutral formalin, at which point they were taken to the pathology department at the University of Washington for paraffin embedding, sectioning, and staining.
Six short axis slices of each muscle were embedded into paraffin blocks. For vascular density determination, three sections from each block were stained for CD-31 (PECAM-1), an endothelial specific antigen, and counterstained with hematoxylin. Digital images of 10 high power fields within each section were obtained, and the ratio of PECAM-1 positive vessels to myofibers was quantified for each field.
For determination of cellular proliferation, 3 sections from each block were stained for Ki-67, a nuclear antigen expressed only in proliferating cells. Digital images of 10 high power fields within each section were obtained, and the number of Ki-67 positive nuclei per high power field were determined.
All data presented are expressed as the mean + SEM, and all groups were compared using a one way ANOVA with a Bonferroni post hoc test. P < 0.05 was considered statistically significant.
Panel D of Figure 1 illustrates the effect of i.v. administration of Na2S on blood flow recovery in the ischemic hind limb. Sodium sulfide administered at 1.0 mg/kg twice daily significantly elevated blood flow in the ischemic limb by day 7 compared to vehicle treatment and was maintained through day 14. These data are consistent with published results from Bir et al. demonstrating an enhancement of blood flow by day 7 in mice administered sodium sulfide at 1.0 mg/kg twice daily .
A second study addressed the questions of dose response and minimal effective dosing of sulfide in the model. To these ends, 4 groups of 7 animals received either 1.0 mg/kg, 0.5 mg/kg, 0.25 mg/kg sodium sulfide, or vehicle twice daily for 14 days following the induction of limb ischemia. Laser doppler measurements of blood flow were taken pre- and post-ligation of the femoral artery on day 0, and then once daily on days 1, 3, 5, 7, 10, 12, and 14. All measurements were made with both the fine needle flow probe used in the pilot study, as well as a miniature surface probe. Hindlimb perfusion was also measured on day 14 by contrast ultrasound.
A final experiment was designed to determine if the changes in blood flow with Na2S treatment persisted after treatment stopped, as well as to attempt to further elucidate a minimum effective dose. To address these questions, 32 animals were divided into 4 groups receiving 0.25 mg/kg sodium sulfide twice daily for 7 days (n = 8), 0.25 mg/kg twice daily for 3 days (n = 8), 0.5 mg/kg once a day for 7 days (n = 8), or vehicle twice daily for 7 days (n = 8). For this experiment, LD blood flow measurements were made pre- and post- ligation on day 0, and then on days 7, 14, and 28. Contrast ultrasound measurements were made on days 14 and 28.
In previous reports of the efficacy of sulfide in the hindlimb model, investigators utilized extended treatment periods. Wang et al. treated rats once daily for 4 weeks, while Bir et al. treated mice twice daily for 2 weeks. In the present study, we started with the dosing regimen used by Bir et al. and incrementally decreased the dosing concentration and frequency until we approached an MED. In our hands, we find that sodium sulfide administered intravenously at 0.25 mg/kg twice daily, or at 0.5 mg/kg once daily, for 7 days was sufficient to restore perfusion to ischemic skeletal muscle. Furthermore, this MED was also shown to provide a prolonged effect that was demonstrable at day 28.
In considering techniques for measuring tissue perfusion, intravenous injection of fluorescent microspheres is a cost effective approach, but it is an end point measurement; lacking the capacity for multiple measurements in a single animal. Laser doppler flowmetry affords the ability to noninvasively measure blood flow in the same animal repeatedly. However, shallow penetration allows only very superficial blood flow to be measured, and occlusion of subdermal microvascular blood flow with the probe itself is a concern with non-scanning probes like those used in this study. We therefore used contrast enhanced ultrasound in conjunction with laser doppler measurements. Ultrasound provides an actual image of the tissue that perfusion is being measured in, offers a much better depth of measurement, and circumvents issues with superficial microvascular occlusion. Results of contrast ultrasound perfusion measurements corroborated those using LD flowmetry.
Clinical trials of therapeutic angiogenesis for PAD and CLI have focused on delivery of constructs encoding genes for proangiogenic growth factors like VEGF, FGF, and HGF or certain bone marrow derived progenitor cell populations, with mixed results. The small size of hydrogen sulfide gas, along with its partition coefficient and rapid diffusion in and through lipid membranes [29,30], can facilitate its delivery to ischemic tissue despite a diminished or absent blood flow. At physiologic pH (7.4), sulfide exists predominantly as the more hydrophilic hydrosulfide anion (HS−), which would be subject to partitioning and is more sensitive to oxidation relative to its fully protonated counterpart. Sulfide becomes increasingly protonated as pH falls, reaching its pKa at 7.0, and then exists predominantly as H2S gas at more acidic pH. The acidification of ischemic tissue would only favor sulfide protonation, which should facilitate its delivery within ischemic tissue.
The results of this study corroborate findings from other labs demonstrating that hydrogen sulfide enhances blood flow and vascular density in the ischemic rat hindlimb when administered at 0.5 and 1.0 mg/kg twice daily over 14 days. The minimum efficacious dose is either 0.25 mg/kg twice daily or 0.5 mg/kg once daily for 7 days. Shortening the treatment window to 3 days was noneffective. Furthermore, these effects appear to be long-lived. These data, together with published results from a number of other labs, lend further credence to the notion that hydrogen sulfide can enhance the vascularity and perfusion of ischemic tissue.
Peripheral arterial disease
Critical limb ischemia
Minimum effective dose
Reactive oxygen species
Vascular endothelial growth factor
Hypoxia inducible factor 1 alpha
Vascular endothelial growth factor receptor 2
Jugular vein catheter
Platelet endothelial cell adhesion molecule 1
Fibroblast growth factor
Hepatocyte growth factor
Sodium hydrogen sulfide
Region of interest
We would like to thank the UW pathology department for embedding, sectioning, and staining of gastrocnemius tissue.
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