Assessing changes in vascular permeability in a hamster model of viral hemorrhagic fever

Abstract

Background: A number of RNA viruses cause viral hemorrhagic fever (VHF), in which proinflammatory mediators released from infected cells induce increased permeability of the endothelial lining of blood vessels, leading to loss of plasma volume, hypotension, multi-organ failure, shock and death. The optimal treatment of VHF should therefore include both the use of antiviral drugs to inhibit viral replication and measures to prevent or correct changes in vascular function. Although rodent models have been used to evaluate treatments for increased vascular permeability (VP) in bacterial sepsis, such studies have not been performed for VHF.

Results: Here, we use an established model of Pichinde virus infection of hamsters to demonstrate how changes in VP can be detected by intravenous infusion of Evans blue dye (EBD), and compare those measurements to changes in hematocrit, serum albumin concentration and serum levels of proinflammatory mediators. We show that EBD injected into sick animals in the late stage of infection is rapidly sequestered in the viscera, while in healthy animals it remains within the plasma, causing the skin to turn a marked blue color. This test could be used in live animals to detect increased VP and to assess the ability of antiviral drugs and vasoactive compounds to prevent its onset. Finally, we describe a multiplexed assay to measure levels of serum factors during the course of Pichinde arenavirus infection and demonstrate that viremia and subsequent increase in white blood cell counts precede the elaboration of inflammatory mediators, which is followed by increased VP and death.

Conclusions: This level of model characterization is essential to the evaluation of novel interventions designed to control the effects of virus-induced hypercytokinemia on host vascular function in VHF, which could lead to improved survival.

Figures

Figure 1

Evidence of increased vascular permeability during late-stage PICV infection in hamsters. A) Two groups of animals (n = 6/group) were infected with ~5 plaque-forming units of PICV or sham (MEM vehicle). On day 7 of infection, EBD-PBS was injected retro-orbitally and the ratio of EBD concentration in the kidney, liver, lung and spleen to the serum EBD concentration was evaluated. Data are expressed as the ratio of absorbance/g of tissue:absorbance of the diluted matching serum sample. *P < 0.05, **P < 0.01 compared to sham-infected hamsters by the Mann-Whitney test (two-tailed). B) Images taken of kidneys, livers (single lobes), lungs, and spleens harvested from a sham-infected control and a PICV-infected hamster on day 7. The serum concentration of EBD was 34% greater in the sham-infected control compared to the infected animal. On the right, images of the respective hamsters' mouths and front paws are included to show the stronger blue coloration notable in the uninfected animal. Dye was extracted from the kidneys, livers, lungs, and spleens shown and the EBD content data are represented in the associated graph.

Figure 2

Analysis of vascular permeability during PICV infection in hamsters. Groups of animals were infected with ~5 plaque-forming units of PICV, with the exception of 4 hamsters that were processed at the time of infection to establish the day 0 baseline reading. On each day of the infection (days 1-8), a group of animals was injected retro-orbitally with EBD-PBS and vascular leak into the (A) liver, (B) spleen, (C) kidney, and (D) lung tissues was evaluated. Data are expressed as the ratio of absorbance/g of tissue:absorbance of the diluted matching serum sample. For days 1-6, there were 5 hamsters/group. Days 7 and 8 had 6 animals/group, but due to the death of one animal in each group prior to time of sacrifice, the analysis is based on 5 hamsters for these groups. *P < 0.05, **P < 0.01 compared to day 0 animals using one-way analysis of variance (ANOVA) with the Newman-Keuls multiple comparison test.

Figure 3

Longitudinal hematologic analysis of PICV infection in hamsters. Two groups of hamsters were infected with ~5 plaque-forming units of PICV or sham-infected. On each of days 0, 2, and 4-8 of the infection, blood was collected by retro-orbital venous sinus route following isoflurane anesthesia for hematologic analysis as described in the methods section. The data points represent the analysis of samples from groups of 4 hamsters with the exception of the PICV-infected group, wherein due to the death of a single animal in the on day 7, and a second hamster on day 8, the data reflect values for 3 and 2 animals, respectively, for days 7 and 8. (A) White blood cells, WBC, (B) hematocrit, HCT, (C) red blood cells, RBC, and (D) hemoglobin, Hb values are shown. m/mm3, millions per cubic milliliter of blood.

Figure 4

Serum albumin levels during the course of PICV infection in hamsters. Groups of hamsters were infected with ~5 plaque-forming units of PICV and serum was obtained following sacrifice on days 1, and 3-7 of PICV infection. Samples were assayed for albumin concentration. Data represent the means and standard deviations of 3-4 hamsters per group. *P < 0.05, **P < 0.01 compared to day 0 animals using ANOVA with the Newman-Keuls multiple comparison test.

Figure 5

Analysis of systemic levels of inflammatory mediators during PICV infection in hamsters. Groups of animals (n = 3-4/group) were infected with ~5 plaque-forming units of PICV, with the exception of 3 hamsters that were processed at the time of infection to establish the day 0 baseline reading. On each of days 2, and 4-7 of the infection, serum was collected from PICV-infected hamsters for multi-analyte profiling of serum antigens. Data are shown for hamster cytokines and chemokines that were sufficiently cross-reactive with the Rodent MAP® detection platform and changed significantly over the course of infection. The least detectable dose is indicated by the red hashed line and is defined in the methods. GCP, Granulocyte Chemotactic Protein; IL, Interleukin; IP, Inducible Protein; MCP, Monocyte Chemoattractant Protein; M-CSF, Macrophage-Colony Stimulating Factor; MDC, Macrophage-Derived Chemokine; MIP, Macrophage Inflammatory Protein; SCF, Stem Cell Factor; VCAM, Vascular Cell Adhesion Molecule; VEGF, Vascular Endothelial Cell Growth Factor.

Figure 6

Analysis of vascular permeability and HCT during YFV infection in hamsters. Groups of animals were infected with ~20 cell culture infectious doses (CCID50) of YFV or sham-infected. On specified days of the infection (days 3-7), groups of YFV-infected hamsters (n = 5) and groups of sham-infected animals (n = 3) were injected retro-orbitally with EBD following collection of whole blood for evaluation of HCT. Vascular leak into the (A) liver, (B) spleen, (C) kidney, and (D) small intestine tissues, and (E) HCT were evaluated. Vascular permeability data are expressed as the ratio of absorbance/g of tissue:absorbance of the diluted matching serum sample. SI, sham-infected. *P < 0.05, ***P < 0.001 compared to the pool of sham-infected animals using ANOVA with the Newman-Keuls multiple comparison test.

Figure 7

Natural history of disease in hamster PICV infection model. The schematic shown is a summary of the present data integrated with previously published findings [12]. Two days after challenge, the presence of PICV in the liver, kidney, spleen, and lung is evident. On day 3, the type I IFN response is measurable and fluctuates slightly over the duration of the acute infection. On days 4 and 5, a significant drop in serum albumin and the first signs of systemic PICV burden, respectively, are observed. By day 6, infectious PICV can be detected in the brain, ALT and AST enzyme levels rise, and WBC and proinflammatory mediators dramatically increase. Day 7 marks the onset of vascular leak, peak viral titer, ALT, and AST concentrations, and initial signs of weight loss. Eight days after challenge, significant vascular leakage is present in multiple tissues, with some animals beginning to succumb and showing clear signs of illness. The majority of the hamsters will succumb by day 9 of infection.