Neural influence on cold induced vasodilatation using a new set-up for bilateral measurement in the rat hind limb

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Abstract

Cold induced vasoconstriction (CIVC) is a way for mammals to reduce heat loss in an effort to maintain body core temperature. As blood flow to a cooled extremity is reduced, the amount of body heat lost at the cooled location is minimised. However, when the extremity temperature gets below a certain threshold, Cold induced vasodilatation (CIVD) occurs, a phenomenon that is believed to reduce the risk of local cold injuries.

Many theories explaining the mechanism of the CIVD reaction have been postulated, but no consensus has been found. One of these theories is that the CIVD reaction is controlled neurally. To study the effect of neural influence on the vascularisation and rewarming patterns a new experimental set-up was designed. This set-up is able to measure responses in both hind paws simultaneously, creating the opportunity to study the effect of nerve injury on one limb and use the contralateral limb as a control.

Ten rats received a sciatic nerve transection and repair of either the left (n = 5) or the right (n = 5) hind limb. Measurements were performed, 1 day pre-operatively, directly post-operatively, and at days 1, 7, 14, 21, 35 and 49 post-operatively.

Although results are not significant, there is a tendency for the CIVD reaction to be reduced in the nerve injured paw until the nerve is regenerated around day 35.

Further investigation of neural influence on the CIVD reaction will be necessary; this set-up may prove to be useful in future experiments to elucidate the mechanism of the CIVD reaction.

Research highlights

▶ Bilateral measurement creates the opportunity to have a within subject control. ▶ There seems to be neural influence on the CIVD reaction. ▶ Cooling with peltier elements creates an opportunity for more accurate CIVD measurement.

Introduction

Cold induced vasoconstriction (CIVC) is a way for mammals to reduce heat loss in an effort to maintain body core temperature. Blood flow to, for example a cooled extremity is reduced, therefore the amount of body heat lost at the cooled location is minimised. However, when extremity temperature gets below a certain threshold, in humans approximately 10 °C, cold induced vasodilatation (CIVD) occurs, a phenomenon that is believed to reduce the risk of local cold injuries such as frostbite (Brown and Baust, 1980, Daanen, 2003, Garcia et al., 2001).

Several hypotheses on the mechanisms responsible for the CIVD reaction are described in the literature. Two hypotheses prevail: (i) the axon reflex theory and (ii) decreased release of norepinephrine: (i) The axon reflex theory states that the cold stimuli excites the receptive unmyelinated nerve endings, probably via C-fibres (Ji et al., 2007), these impulses are transmitted centrally and via the axon branches. The sensory nerve endings then release vasoactive substances resulting in vasodilatation (Hornyak et al., 1990). (ii) The decreased release of norepinephrine; the sensitivity of the norepinephrine receptors increases in the cold. This results in a reduced blood flow and therefore a further decrease in tissue temperature. This in time may result in a reduction of adrenergic neurotransmission, such as norepinephrine due to the cold and this will, as a consequence, increase the blood flow (Daanen, 2003, Gardner and Webb, 1986, Ji et al., 2007).

Furthermore, several blood flow, vascular impedance and temperature measurement methods have been used, such as Laser-Doppler flowmetry, strain gauge plethysmography and skin temperature measurement. However skin temperature measurement is the most commonly used. In this study thermocouples are attached to the skin to measure the CIVD reaction in mammals (Daanen, 2003). A regularly used method to provoke a CIVD reaction is immersion of the limb or tail in stirred ice water. If a thermocouple is then attached to this immerged body part, temperature data will be an average of body part temperature and the stirred ice water. Therefore, a different method had to be developed to eliminate this temperature measurement bias. In this new set-up, modified peltier cooling elements were used to cool the ventral side of the hind paw, and skin temperature was measured on the dorsal side of the paw, thereby excluding influence of the cooling medium on the data. To be able to study the effect of nerve damage on the CIVD reaction a within subject design study was chosen. Whereas most papers describe a method for unilateral paw or tail cooling (Brown and Baust, 1980, Hellon and Taylor, 1982, Hellstrom, 1975, Owens et al., 2002, Thomas et al., 1994), the measurement set-up developed for this paper is able to trigger a CIVD reaction in both hind paws simultaneously. Therefore a comparison between hind paws with and without nerve damage within the same subject can be obtained, eliminating factors influencing the CIVD reaction that change in time, for instance ambient temperatures.

In our hypothesis a CIVD reaction only occurs when there is an intact nerve innervating the cooled area. This hypothesis is enhanced by the thought that CIVC needs to occur before CIVD, and as there is no CIVC in uncooled parts of the body, no CIVD will take place. It is therefore assumed that the CIVD reaction only occurs in the cooled extremity and is not a generalised reaction. There are many factors influencing CIVD, such as body core temperature (Flouris et al., 2008), ambient temperature, gender (Daanen, 2003), age (Spurr et al., 1955) and stress (Daanen, 2003) therefore, considerable variations in the CIVD reaction within and between subjects can be expected. The aim of this study is to create a newly developed set-up that provides simultaneous bilateral assessment of the CIVD following nerve injury, also minimising the influence of external factors such as cooling medium temperature.

Section snippets

Methods

A well-described method is to submerge the extremity in stirred ice water. However, submergement influences direct thermal measurement of the skin by thermocouples by averaging skin temperature and ice water temperature. A new set-up was designed to measure the CIVD response bilaterally.

Results

Figs. 2.1–2.8 show the time course of temperature measurements of rat number 2 at the day before surgery, immediately post-operative (day 0) and at day 1, 7, 14, 21, 35 and 49 post-operative. The temperature of both the left and right paw rapidly declined caused by the cooling of the paws by the peltier elements. The paw temperature dropped to about 10 °C, followed by multiple CIVD responses. The lines show distal paw temperature and temperature recordings of the peltier elements.

Discussion

This was the first study to evaluate CIVD responses in both hind limbs in a rat. By using this new measurement set-up, we were able to obtain results showing different CIVD responses in both limbs. Other studies concerning CIVD responses in a rat mostly use set-ups to show unilateral responses or responses in the tail (Brown and Baust, 1980, Hellstrom, 1975, Thomas et al., 1994), whereas this study now facilitates investigating the mechanism behind the CIVD response; the ability to inflict

Acknowledgement

The authors are grateful to Mrs Ineke Hekking for her assistance in surgery and with the measurements.

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