Homocysteine-induced peripheral microcirculation dysfunction in zebrafish and its attenuation by L-arginine

Abstract

Elevated blood homocysteine (Hcy) level is frequently observed in aged individuals and those with age-related vascular diseases. However, its effect on peripheral microcirculation is still not fully understood. Using in vivo zebrafish model, the degree of Hcy-induced peripheral microcirculation dysfunction is assessed in this study with a proposed dimensionless velocity parameter [Formula: see text], where [Formula: see text] and [Formula: see text] represent the peripheral microcirculation perfusion and the systemic perfusion levels, respectively. The ratio of the peripheral microcirculation perfusion to the systemic perfusion is largely decreased due to peripheral accumulation of neutrophils, while the systemic perfusion is relatively preserved by increased blood supply from subintestinal vein. Pretreatment with L-arginine attenuates the effects of Hcy on peripheral microcirculation and reduces the peripheral accumulation of neutrophils. Given its convenience, high reproducibility of the observation site, non-invasiveness, and the ease of drug treatment, the present zebrafish model with the proposed parameters will be used as a useful drug screening platform for investigating the pathophysiology of Hcy-induced microvascular diseases.

Keywords: L-arginine; homocysteine; inflammation; peripheral microcirculation dysfunction; zebrafish.

Figure 1. Schematics of the vascular network of a 10 dpf zebrafish

Vessels are composed of the dorsal artery (DA), posterior cardinal vein (PCV), caudal vein (CV), and subintestinal vein (SIV). Each region of interest (ROI) is represented by dotted box. The time-averaged RBC velocity and peak systolic RBC velocity in the DA are defined as ${\overline{\text{V}}}_{\text{ADA}}$ and ${\widehat{\text{V}}}_{\text{PDA}}$, respectively. The time-averaged RBC velocities in the PCV and CV regions are defined as ${\overline{\text{V}}}_{\text{PCV}}$ and ${\overline{\text{V}}}_{\text{CV}}$, respectively.

Figure 2. Hcy-induced PMD reduces blood flow in the CV

The minimum concentration of 50 μM Hcy greatly reduces (A) ${\overline{\text{V}}}_{\text{CV}}/{\overline{\text{V}}}_{\text{PCV}}$. Meanwhile, the highest concentration of 500 μM Hcy significantly reduces (B) ${\overline{\text{V}}}_{\text{CV}}/{\overline{\text{V}}}_{\text{ADA}}$. However, (C) ${\overline{\text{V}}}_{\text{PCV}}/{\overline{\text{V}}}_{\text{PDA}}$ and (D) ${\overline{\text{V}}}_{\text{PCV}}/{\widehat{\text{V}}}_{\text{PDA}}$ do not noticeably change, indicating that systemic perfusion is relatively preserved. Asterisk (*) denotes significant decrease in peripheral microcirculation perfusion according to the level of Hcy-induced PMD, compared with the control. n=4-6 per Hcy group; * p<0.05 (one-way ANOVA with Newman-Keuls's post-test). Resulting values are expressed as mean ± SD (standard deviation).

Figure 3. Variation in the number ratio of RBCs in the SIV and PCV regions

The number ratio of RBCs passing through the SIV (RBC_SIV) relative to the PCV (RBC_PCV) is increased with the elevation of Hcy level for maintaining systemic perfusion against Hcy-induced PMD in the tail region. Asterisk (*) denotes significant increase in compensatory blood flow from the SIV. n = 4-6 per Hcy group; * p<0.05 (one-way ANOVA with Newman-Keuls's post-test). Resulting values are expressed as mean ± SD (standard deviation).

Figure 4. Pretreatment with 50 μM L-arginine attenuates the onset of Hcy-induced PMD

The degree of PMD is assessed with the non-dimensional parameter ${\overline{\text{V}}}_{\text{CV}}/{\overline{\text{V}}}_{\text{PCV}}$. Asterisk (*) denotes the significant protective effect of L-arginine pretreatment against Hcy-induced PMD. n = 7–13 per group; ** p<0.01 and *** p<0.001 (one-way ANOVA with Newman-Keuls's post-test). L-arg indicates L-arginine. Resulting values are expressed as mean ± SD (standard deviation).

Figure 5. Accumulation of neutrophils in the tail region

The stained neutrophils in the tail region of (A) control, (B) the 100 μM Hcy-exposed, and (C) the 50 μM L-arginine pretreated 100 μM Hcy-exposed zebrafish are visualized. Arrows indicate significantly aggregated neutrophils. (D) The areas with accumulated neutrophils in the tail region are quantitatively compared. n = 5–12 per group; **p<0.01 and *** p<0.001 (one-way ANOVA with Newman-Keuls's post-test). Control, control zebrafish without any treatment; Hcy, Hcy-exposed zebrafish; L-arginine + Hcy, L-arginine pretreated Hcy-exposed zebrafish. Scale bar denotes 100 μm.