Evaluation of Postocclusive Reactive Hyperemia in Rat Paw Skin Using Laser Speckle Contrast Imaging

Summary

This protocol presents a method for assessing parameters of postocclusive reactive hyperemia in the skin of an anesthetized rat using laser speckle contrast imaging and invasive blood pressure measurement.

Abstract

Laser Speckle Contrast Imaging (LSCI) is a modern, non-invasive technique for assessing microvascular perfusion in humans and laboratory animals. The combination of LSCI with invasive blood pressure (BP) measurement and a vascular occlusion test allows the study of postocclusive reactive hyperemia (PORH), providing an assessment of microvascular blood flow reserve and the vascular reactivity. This article demonstrates the technique of using the LSCI to record and visualize PORH in the hind paw of a rat under general anesthesia. A special pneumatic cuff is inflated to create a temporary (180 s) vascular occlusion at the level of the tibia. Evaluation of the subsequent PORH (peak hyperemia and kinetic indices) can provide additional information on the mechanisms and severity of the pathological process under study (circulatory shock, heart failure, hypertension, etc.) or identify vascular effects of a new drug that are not apparent when assessing perfusion at rest. Invasive BP monitoring allows calculation of cutaneous vascular conductance and consideration of changes in systemic hemodynamics, which is particularly important in experimental models of critical illness. The use of this approach to identify microvascular dysfunction in a rat model of hemorrhagic shock will also be demonstrated.

Introduction

Microvascular dysfunction is involved in the pathogenesis of many cardiovascular, endocrine and other diseases, compromising tissue perfusion and oxygenation, and disrupting the vasomotor regulatory mechanisms of systemic hemodynamics (for example, in hypertension)¹. Microcirculation disorders also have great pathophysiological significance in the mechanisms of organ dysfunction in critically ill patients with trauma, sepsis and various types of shock (hemorrhagic, septic, cardiogenic, etc.)².

Currently, several laser-based methods are used in clinical and preclinical studies for non-invasive ....

Protocol

​The procedures described below were performed as part of a protocol approved by the Ethical Review Board of the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology.

1. Preparation of the pneumatic cuff for vascular occlusion

1. Use a pneumatic cuff from a noninvasive blood pressure (NIBP) measurement system for rats to create transient vascular occlusion on the hind paw of a rat.
2. Connect the cuff to the aneroid manometer and the rubber pump of a conventional mechanical tonometer for measuring blood pressure in humans. Use three segments of transparent 3 mm plastic tubing (1 m of which is in....

Results

The above-described method for assessing microvascular function was applied by us in a series of experiments with modeling of hemorrhagic shock (blood loss of 35% of the calculated blood volume) in Wistar male rats weighing 250-300 g. In the group of animals with hemorrhage (HS, n = 13), the assessment of PORH parameters with simultaneous monitoring of invasive BP was performed 30 min after the completion of hemorrhage. In the control group of sham-operated animals (Sham, n = 10), hemorrh.......

Discussion

Here we describe a modernized technology for assessing cutaneous microcirculation and vascular reactivity in an in vivo experiment in rodents. For successful and standardized use of this method in physiological and pharmacological studies, several additional considerations should be taken into account. In the above protocol, rats are anesthetized with a combination of tiletamine + zolazepam + xylazine, which is dictated mainly by local standards for working with laboratory animals and limited access to buprenorphine, ket.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Atraumatic needle with surgical suture nylon, 4C-0.5x16-4/0-N	Medtekhnika, Russia	N/A	Blue. Length 75 cm. USP 4/0 (MP1.5). For suturing a surgical wound
Catheter for rat's carotid artery with blunt-end syringe needle 22 G	SciCat, Russia	N/A	PE-50, length 12 cm, OD=0.9 mm, ID= 0.6 mm.With one fixing ring
Deltran Disposable Pressure Transducer	Utah Medical Products, US	6069	12" cable
DiscofixC 3-way stopcock for infusion therapy and monitoring	B. Braun, Germany	16494C
Heparin	Belmedpreparaty, Belarus	N/A	Heparin sodium 5000 IU/ml, ampoules 5 mL
Lidocaine	Velpharm, Russia	N/A	Solution for injection 20 mg/mL, ampoules 2 mL
Pneumatic cuff "Systole"	Neurobotics, Russia	N/A	Part of non-invasive blood pressure measurement for rodents. Type 5-10 mm tail (rats)
Sodium chloride	Pharmasyntez, Russia	N/A	Solution for infusion 0,9%, 250 mL
Software LabChart8	ADInstruments, Australia	N/A	Version 8.1.13
Software MATLAB	The MathWorks Inc., US	N/A	Version R2019b
Sterile syringe for single use, 1 mL, 3-part	Huaian City Hengchun Medical Product Co. Ltd., China	N/A	27 G needle
Sterile syringe for single use, 2 (2.5) mL, 3-part	Vogt Medical Vertrieb GmbH, Germany	1310524	23 G needle
Suture SILK-S	Polytechmed, Russia	N/A	Non-absorbable, braided,made of natural silk with a silicone coating, black in cassette. USP 4/0 (MP1.5). Length 100 m. For ligating vessels
Xyla	Interchemie, Netherlands	N/A	Xylazine 20 mg/mL, 50 mL
Zoletil 100	Virbac, France	N/A	Tiletamine HCl 50 mg/mL, zolazepam HCl 50 mg/mL, 5 mL