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(Stroke. 2002;33:256.)
© 2002 American Heart Association, Inc.

Original Contributions

Cooling-Induced Carotid Artery Dilatation

An Experimental Study in Isolated Vessels

Seham Mustafa, PhD Olav Thulesius, MD, PhD

From the Department of Pharmacology and Toxicology, Faculty of Medicine, Kuwait University, Safat, Kuwait.

Correspondence to Dr Seham Mustafa, Department of Pharmacology and Toxicology, Faculty of Medicine, PO Box 24923, Safat 13110, Kuwait. E-mail seham{at}hsc.kuniv.edu.kw

	Abstract

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Background and Purpose— Clinical and experimental studies seem to indicate that hypothermia may improve outcome in stroke victims and reduce experimental brain injury. The current interpretation is that cooling has a neuroprotective effect by reducing brain metabolism. The objective of our study was to test the hypothesis that hypothermia induces arterial vasodilatation and thereby increases cerebral blood flow.

Methods— We recorded isometric tension in rabbit carotid artery strips in organ baths during stepwise cooling. The cooling responses were tested at basal tone, in noradrenaline-precontracted vessels, and after electric field stimulation.

Results— Stepwise cooling from 37°C to 4°C induced reproducible graded relaxation, inversely proportional to temperature. The responses could be elicited at basal tone and in precontracted vessels. Cooling decreased the contractile responses to norepinephrine and potassium chloride. Cooling at 20°C decreased the contractile responses to electric field stimulation, while at 10°C these were totally abolished. Cooling-induced vasodilatation is not dependent on an endothelial mechanism.

Conclusions— Cooling of carotid artery preparations induced a reversible graded vasodilatation and decreased or abolished the effect of vasocontractile neurotransmitters. The effect of local hypothermia could increase cerebral blood flow and may constitute a positive therapeutic modality in stroke patients.

Key Words: carotid arteries • hypothermia • ischemia • neuroprotection • stroke

	Introduction

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We previously studied thermal responses of smooth muscle in various species and organs of the body and found that the degree of active tension is very temperature dependent. Moreover, we detected marked regional differences in the cooling-response pattern in superficial and deep blood vessels, trachea, bronchi, urinary bladder detrusor muscle, and gut. Cutaneous veins contract, whereas deep extremity vessels dilate.¹ The aorta and pulmonary artery clearly show a cooling-induced relaxation,² whereas airways,³ urinary bladder,⁴ and gut contract.⁵

As a treatment, hypothermia has been advocated as a potent modality in protecting neurons against lethal ischemic stress. In animal experiments, intraischemic hypothermia has been shown to be neuroprotective in global⁶ and focal^7–9 cerebral ischemia and even when hypothermia is initiated after temporary cerebral ischemia.¹⁰ Kuluz et al¹¹ found that selective brain cooling increases cortical cerebral blood flow.

It has been claimed that mild hypothermia is possibly the single most effective method of cerebroprotection developed to date.¹² Krieger et al¹³ recently reported that hypothermia is effective in patients with acute brain infarction, demonstrating that hypothermia appears feasible and safe in cases of acute ischemic stroke even after thrombolysis. However, many questions regarding hypothermia remain to be addressed, and recently some clinical studies have questioned the effectiveness of hypothermia for traumatic brain injury.¹⁴ In view of these considerations and the positive reports about hypothermia treatment in stroke victims,¹⁵ we decided to extend our studies to include the carotid artery to test the hypothesis of a cooling-induced vasodilatation of brain supply vessels.

	Materials and Methods

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All procedures followed were within institutional guidelines. Adult male New Zealand White rabbits weighing 2.5 to 3.5 kg were anesthetized with pentobarbital (120 mg/kg IP). The carotid arteries were removed and dissected free of extraneous fat and connective tissue and placed in Krebs’ solution of the following composition (mmol/L): NaCl 118, KCl 5.9, MgSO₄ 1.2, CaCl₂ 2.2, KH₂PO₄ 1.2, NaHCO₃ 26, glucose 11.1, pH 7.4. The arteries were cut into 5-mm ring segments that were mounted on triangular wire supports and suspended in 10-mL organ baths containing Krebs’ solution, maintained at 37°C, and gassed with 95% O₂ and 5% CO₂. Tension was continuously recorded with the use of a computerized, automated isometric transducer system (Schuler organ bath 809; Hugo Sachs Electronik) connected to a Gould recorder. The segments were initially loaded to a tension of 2 g and allowed to equilibrate for 60 minutes, during which time they were washed twice. Care was taken not to injure the endothelium during the preparation. The presence of intact endothelium was verified by adding acetylcholine (1 µmol/L), which resulted in relaxation of norepinephrine (1 µmol/L)–precontracted rings. At the end of each experiment the muscle was weighed, and responses were calculated as milligram per milligram tissue weight or percent change from norepinephrine-induced condition.

Cooling Protocol
The organ bath temperature was reduced with the use of a cooling circulator bath (Haake F3, Fisons), which had been set to the appropriate temperature. It took 2 to 3 minutes to reach the desired temperature in steps from 37°C to 4°C (37°C, 30°C, 25°C, 20°C, 15°C, 10°C, 4°C). Each cooling period was maintained until a peak response had leveled off before the temperature was reduced further.

Electric Field Stimulation
For electric field stimulation (EFS), tissues were suspended between 2 platinum plate electrodes. The electrodes were connected to a Grass S8800 stimulator, delivering square wave pulses. Previously determined optimum electric stimulation parameters (70 V, 0.5 ms for 15 seconds with a frequency ranging from 2.5 to 40 Hz) were used.

Drugs
Norepinephrine hydrochloride, acetylcholine hydrochloride, sodium nitroprusside, phentolamine hydrochloride, cocaine, propranolol hydrochloride, EGTA, and tetrodotoxin were obtained from Sigma Chemical Co. All drugs were dissolved in distilled water except EGTA, which was dissolved in 0.1 NaOH.

Statistical Analysis
Data are presented as mean (SEM). When necessary, differences between 2 mean values were compared by Student’s t test (paired or unpaired as appropriate). When multiple comparisons were necessary, 1-way ANOVA was used, followed by Student-Newman-Keuls test. The difference was assumed to be significant at P<0.05.

	Results

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Cooling-Induced Relaxation
A typical original tracing representing cooling-induced relaxation of rabbit carotid artery specimens from basal tone and after induction of precontraction with 1 µmol/L norepinephrine is shown in Figure 1. Before cooling, all preparations maintained a stable baseline. Lowering of the bath temperature induced a rapid and reproducible stepwise decrease in tone from either the basal level or norepinephrine-induced tone, inversely proportional to the temperature. Maximum relaxation was achieved at a temperature of 4°C (Figure 2). When the temperature was reset to 37°C, the tone rapidly returned to the basal level.

View larger version (12K): [in this window] [in a new window]   Figure 1. Original recording of stepwise graded cooling and rewarming of isolated carotid arterial smooth muscle strips of rabbits, showing basal tone (A) and norepinephrine (NE) (1 µmol/L)–induced contraction (B).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of cooling on basal tone (top) and norepinephrine (NE) (1 µmol/L)–induced contraction (bottom) in isolated carotid arterial smooth muscle strips of rabbits. Each point represents mean±SEM of 5 experiments.

Cooling to 15°C relaxed the norepinephrine-induced contraction to the basal level as sodium nitroprusside and acetylcholine in the presence of an intact endothelium, but cooling also relaxed carotid artery strips without endothelium.

Cooling and Vasoconstrictor Drugs
Norepinephrine (1 nmol/L to 100 µmol/L) and KCl (3 to 24 mmol/L) induced concentration-dependent contractions. Dose-response curves for norepinephrine and KCl were obtained at 37°C, 20°C, and 10°C. Cooling to 20°C significantly inhibited norepinephrine- and KCl-induced contractions, and a further reduction of temperature to 10°C abolished contractile responses (Figure 3). Cooling also lengthened the time to the peak tension and doubled the time required to obtain a dose-response curve for both norepinephrine and KCl.

View larger version (21K): [in this window] [in a new window]   Figure 3. Influence of cooling on norepinephrine and KCl concentration-contraction curves at 37°C, 20°C, and 10°C in isolated carotid arterial smooth muscle strips of rabbits. Each point represents mean±SEM of 5 experiments. *P<0.05.

The incubation of carotid artery in calcium-free, EGTA (0.2 mM)-containing Krebs’ solution for 30 minutes did not change the basal tension, did not change the level of basal tone, and did not affect the responses to norepinephrine, but it abolished the contractile response to KCl.

EFS at 37°C
EFS (2.5 to 40 Hz) evoked frequency-dependent contractions that were rapid in onset (Figure 4). The contractions were reproducible, and there was no evidence of tachyphylaxis in any of the preparations tested. Tetrodotoxin (1 µmol/L; n=8; data not shown) abolished these contractions, indicating that they were neurogenic. The contractions were also abolished by phentolamine (1 µmol/L) in all cases, indicating that the excitatory innervation of carotid artery is adrenergic in origin. Propranolol (1 µmol/L) did not increase the responses to EFS, indicating the absence of ß-adrenergic receptors in the rabbit carotid artery.

View larger version (11K): [in this window] [in a new window]   Figure 4. Original recording of frequency-contraction responses to EFS (70 V, 0.5 ms, 15 seconds) in isolated carotid arterial smooth muscle strips of rabbits at 37°C, 20°C, and 10°C.

Cocaine (3 µmol/L) inhibited the responses to EFS and also lengthened the time required to return to basal tone after stimulation ceased. Contraction started after 10 seconds of stimulation. This response to cocaine indicates that the reuptake-1 is important and that the artery is highly innervated.

Incubation of the carotid artery in a calcium-free, EGTA (0.2 mM)-containing Krebs’ solution for 30 minutes did not change the basal tension but decreased the responses to EFS. The contraction started after 5 seconds of stimulation while using calcium-free, EGTA (2 mM)-containing Krebs’ solution abolished the responses to EFS.

Cooling and EFS
Reducing the bath temperature from 37°C to 20°C resulted in relaxation. When the temperature was maintained at 20°C, EFS induced frequency-dependent contractions of the carotid artery preparations. However, these responses were slower in onset and smaller in amplitude. The contraction started after 10 seconds of stimulation. Further cooling to 10°C induced more pronounced relaxation of basal tone, and EFS no longer elicited any contractile response. Thus, hypothermia reduced the effect of vasoconstriction by sympathetic activation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present investigation clearly shows that hypothermia induces vasodilatation of the carotid artery of rabbits by smooth muscle relaxation. The degree of cooling-induced relaxation of isolated rabbit carotid artery preparations is inversely proportional to temperature. These results confirm our earlier findings showing cooling-induced relaxation obtained in deep extremity arteries of sheep¹ and the aorta and pulmonary artery of rats.²

To establish the cellular mechanism of cooling-induced relaxation, we studied the effect of the vasoconstrictor agents norepinephrine and KCl. Both of these agents induced contraction that was extensively reduced with cooling. Norepinephrine and KCl have a different cellular mechanism of action: norepinephrine acts through phospholipase C, releasing inositol 1,4,5-triphosphate (IP₃), which leads to increased intracellular Ca²⁺, while KCl depolarizes the muscle cell, permitting extracellular Ca²⁺ influx through voltage-dependent Ca²⁺channels. Therefore, the action of norepinephrine depends on intracellular Ca²⁺, and that of KCl depends on entry of extracellular Ca²⁺¹⁶; therefore, the use of a Ca-free solution did not affect the contraction to norepinephrine. In a previous study we showed that hypothermia decreased the influx of Ca²⁺ into the smooth muscle cell¹⁷; therefore, cooling had a more pronounced effect on KCl than norepinephrine and EFS.

We also studied the neurogenic consequences of hypothermia by monitoring the effect of sympathetic vasoconstrictor tone induced by EFS of the carotid artery strips. The contractile response to EFS was progressively reduced with decreasing temperature and was totally abolished at 10°C. This means that the release of norepinephrine is temperature dependent and is reduced by hypothermia. In addition to the action of hypothermia on neurogenic adrenergic vasoconstrictor tone, cooling slows all energy-requiring metabolic processes and therefore the process of contraction itself at any level of activation.

While cooling lowers pH of the physiological salt solution, the change in pH of Krebs’ solution over the temperature range of 37°C to 4°C was approximately 0.15 U. It is therefore highly unlikely that this change in pH had any effect on the experimental results. This finding is similar to other reports, indicating that the temperature-induced change in pH cannot account for either the relaxation or contraction responses to cooling.^2,18

Both intracellular alkalinization- and acidosis-induced increases in intracellular Ca⁺² were mediated by both extracellular Ca⁺² influx and release of stored intracellular Ca⁺², leading to contraction in isolated canine and ferret pulmonary arterial smooth muscle^19,20 and rat isolated thoracic aorta.²¹ Our previous study² showed that neither Ca⁺²-free, EGTA (2 mmol/L)–containing Krebs’ solution nor nifedipine and thapsigargin affected cooling-induced relaxation, indicating that cooling-induced relaxation is not affected by the change in intracellular pH.

In contrast to our present findings of cooling-induced carotid artery relaxation, in previous studies we reported totally divergent responses in other smooth muscle–lined organs. In these organs hypothermia induced a clear-cut increase in basal tone and in some organs induced an increase in the frequency of rhythmic contractions. Ovine airways,³ rat bladder detrusor muscle,⁴ and rat gastrointestinal smooth muscle⁵ all contracted when exposed to cooling, contrary to the observed effect in the carotid artery. In these organs, however, pattern of innervation, type of transmitters, release process, and inactivation are different.

Therefore, the basic mechanisms underlying hypothermic reactions of smooth muscle in blood vessels and other conduits of the body are regionally different and adjusted to serve the functional requirements of the organism when exposed to hypothermia. From the functional point of view, it is reasonable to assume that survival during hypothermia requires adjustments in which the priority of organs must be considered. The circulation of blood should be directed toward the central nervous system and the heart, whereas other areas with less demand may be temporarily shut off. A balance of vasoconstriction to prevent heat loss and vasodilatation to secure supply to vital organs can be expected.

From the clinical point of view, our experiments support the view that moderate hypothermia may be useful for maintaining and, if necessary, increasing blood supply to ischemic regions compromised by reduced arterial perfusion. Despite recent reports on the ineffectiveness of generalized hypothermia in the treatment of acute brain injury,¹² our study may point to an alternative solution: instead of generalized hypothermia, local cooling of the arterial supply vessels to the brain (in the neck) may be considered a new possible therapeutic alternative.

The feasibility of induction of carotid artery cooling (of the arterial wall) and clinical use of cooling-induced relaxation as a therapeutic modality needs to be carefully investigated in further controlled studies. This can be done by monitoring carotid artery flow velocity and diameter with ultrasound as well as measuring tympanic temperature. Administration of graded cooling can be achieved by the external application of an ice-water–perfused neck collar. We are presently performing this phase of research.

Our study is the first to suggest carotid artery cooling as a therapeutic procedure. Previous attempts have used venous cooling of facial veins and dural sinuses,²² but our method seems to be more straightforward and is aimed at inducing carotid artery vasodilatation as well as cooling of blood directed to the brain.

	Acknowledgments

 
The authors acknowledge the Faculty of Medicine, Kuwait University, for supplying the animals, chemicals, and equipment for performing this research.

Received June 19, 2001; revision received October 17, 2001; accepted October 22, 2001.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Thulesius O, Yousif MH. Na⁺, K^--ATPase inhibition: a new mechanism for cold-induced vasoconstriction in cutaneous veins. Acta Physiol Scand. 1990; 141: 127–128.

2. Mustafa SMD, Thulesius O. Cooling is a potent vasodilator of deep vessels in the rat. Can J Physiol Pharmacol. 2001; 79: 899–904.[CrossRef][Medline] [Order article via Infotrieve]

3. Mustafa SMD, Pilcher CWT, Williams KI. Cooling-induced contraction in ovine airways smooth muscle. Pharmacol Res. 1999; 39: 113–123.[Medline] [Order article via Infotrieve]

4. Mustafa SM, Thulesius O. Cooling-induced bladder contraction: studies on isolated detrusor muscle preparations in rat. Urology. 1999; 53: 653–657.[Medline] [Order article via Infotrieve]

5. Mustafa SMD, Thulesius O. Cooling induced gastrointestinal contraction in the rat. Fundam Clin Pharmacol. In press.

6. Welsh FA, Sims RE, Harris VA. Mild hypothermia prevents ischemic injury in gerbil hippocampus. J Cereb Blood Flow Metab. 1990; 10: 557–563.[Medline] [Order article via Infotrieve]

7. GotoY, Kassell NF, Hiramatsu K, Soleau S, Lee KS. Effect of intraischemic hypothermia on cerebral damage in a model of reversible focal ischemia. Neurosurgery. 1993; 32: 980–985.[Medline] [Order article via Infotrieve]

8. Morikawa E, Ginsberg MD, Dietrich WD, Duncan RC, Kraydieh S, Globus MY, Busto R. The significance of brain temperature in focal ischemia: histopathological consequences of middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1992; 12: 380–389.[Medline] [Order article via Infotrieve]

9. Ridenour TR, Warner DS, Todd MM, McAllister AC. Mild hypothermia reduces infarct size resulting from temporary but not permanent focal ischemia in rats. Strocke. 1992; 23: 733–738.

10. Yanamoto H, Nagata I, Nakahara I, Tohnai N, Zhang Z, Kikuchi H. Combination of intraischemic and postischemic hypothermia provides potent and persistent neuroprotection against temporary focal ischemia in rats. Stroke. 2000; 30: 2720–2726.[Abstract/Free Full Text]

11. Kuluz JW, Prado R, Chang J, Ginsberg MD, Schleien CL, Busto R. Selective brain cooling increases cortical cerebral blood flow in rats. Am J Physiol. 1993; 265: H824–H827.[Abstract/Free Full Text]

12. Maier CM, Ahern KB, Cheng ML, Lee JE, Yenari MA, Steinberg GK. Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia: effects on neurologic outcome, infarct size, apoptosis, and inflammation. Stroke. 1998; 29: 2171–2180.[Abstract/Free Full Text]

13. Krieger DW, De Georgia MA, Abou-Chebl A, Andrefsky JC, Sila CA, Katzan IL, Mayberg MR, Furlan AJ. Cooling for acute ischemic brain damage (COOL AID): an open pilot study of induced hypothermia in acute ischemic stroke. Stroke. 2001; 32: 1847–1854.[Abstract/Free Full Text]

14. Clifton GL, Miller ER, Choi SC, Levin SM, Smith KR, Muizelaar JP, Wagner FC, Marion DW, Luerssen TG, Chesnut RM, Schwartz M. Lack of effect of induction of hypothermia after acute brain injury. N Engl J Med. 2001; 344: 556–563.[Abstract/Free Full Text]

15. Behringer W, Prueckner S, Kentner R, Tisherman SA, Radovsky A, Clark R, Stezoski W, Henchir J, Klein E, Safar P. Rapid hypothermic aortic flush can achieve survival without damage after 30 minutes cardiac arrest in dogs. Anesthesiology. 2000; 93: 1491–1499.[CrossRef][Medline] [Order article via Infotrieve]

16. Droogmans G, Himpens B, Casteels R. Ca-exchange, Ca-channels, and Ca-antagonists. Experientia. 1985; 41: 895–900.[Medline] [Order article via Infotrieve]

17. Mustafa SMD, Pilcher CWT, Williams KI. Cooling-induced bronchoconstriction: the role of ion-pump and ion-carrier system. Pharmacol Res. 1999; 39: 125–136.[CrossRef][Medline] [Order article via Infotrieve]

18. Park MK, Hayashi S, Robotham J. Cooling-induced contraction of guinea-pig tracheal smooth muscle. Proc Soc Exp Biol Med. 1984; 177: 283–289.[CrossRef][Medline] [Order article via Infotrieve]

19. Krampetz IK, Rhoades RA. Intracellular pH: effect on pulmonary arterial smooth muscle. Am J Physiol. 1991; 260: L516–L521.[Abstract/Free Full Text]

20. Farrukh IS, Hoidal JR, Barry WH. Effect of intracellular pH on ferret pulmonary arterial smooth muscle cell calcium homeostasis and pressure. J Appl Physiol. 1996; 80: 496–505.[Abstract/Free Full Text]

21. Horie S, Yano S, Watanabe K. Intracellular alkalization by NH₄Cl increases cytosolic Ca²⁺ level and tension in the rat aortic smooth muscle. Life Sci. 1995; 56: 1835–1843.[Medline] [Order article via Infotrieve]

22. Caputa M, Demicka A, Dokladny K, Kurowicka B. Anatomical and physiological evidence for efficacious selective brain cooling in rats. J Therm Biol. 1996; 21: 21–28.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mustafa, S. Articles by Thulesius, O. Search for Related Content PubMed PubMed Citation Articles by Mustafa, S. Articles by Thulesius, O. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB POTASSIUM CHLORIDE Related Collections Animal models of human disease Acute Cerebral Infarction Emergency treatment of Stroke Neuroprotectors Other Stroke Treatment - Medical