Vasospasm of the Flap Pedicle – Magnesium Sulphate Relieves Vasospasm of Axial Flap Pedicle in Porcine Model

INTRODUCTION

Vasospasm is a common problem in microvascular surgery. It is a localized contraction of the vascular smooth muscle in contrast to a generalized vasoconstriction, which is caused by influences of central nervous system. Vasospasm usually develops as a result of surgical manipulation of small vessels of the vascular pedicle during free flap elevation. Often, it causes only temporary and incomplete obstruction of the vessels. In some cases, however, a prolonged vasospasm may result in formation of a thrombus and cause a complete obstruction of the vessel.^1-5 Definition of vasospasm, its pathogenesis and clinical consequences is mentioned in the first part of our vasospasm study, which is published as a separate article (Vasospasm of the Flap Pedicle: The New Experimental Model on Rat).

Surgical treatment of acute vasospasm is rarely effective, therefore pharmacologic therapy should be administered. The ideal chemical agent is still being sought.^6-16 The effects of different vasodilating drugs were compared in the second part of our study, which is also published as a separate article (Vasospasm of the Flap Pedicle – The Effect of 11 Most Often Used Vasodilating Drugs-Comparative Study in a Rat Motel). Vasospasm was provoked by tension applied on the pedicle of a groin flap on rat, magnesium sulphate 10% was the most efficient chemical agent among a number of studied drugs. According to our knowledge, the effect of magnesium on the pedicle of the flap in porcine model has not been studied yet.

The purpose of this experimental study was to evaluate the effect of magnesium sulphate on vasospasm provoked by surgical manipulation (axial tension) on the flap pedicle in a pig. Tension on the flap pedicle commonly appears during surgical manipulation of the vessel in clinical practice and this stimulus was evaluated as the most appropriate for drug testing in our previous experiments on rat. This stimulus is well defined and easily repeatable.

MATERIAL AND METHODS

The study was done at Veterinary Research Institute (Brno, Czech Republic), and it was approved by the Ethics Committee of the Ministry of Agriculture of the Czech Republic for the animal studies.

This experiment was done on a porcine model; and it was based on laser-Doppler measurement of the peripheral blood perfusion of the bilateral axial flaps based on the arteria and vena epigastrica caudalis superficialis. The flap on the left side served as the study group (A) and the flap on the right side served as the control group (B). Vasospasm was provoked in both groups by pulling the pedicle in the longitudinal axis of the pedicle vessels. This kind of stimulus could not be studied in a clinical environment without putting flap viability into risk. From this point of view, the animal experimental model was necessary.

Eight pigs, crossbreeds of White Noble (50%) and Landrace (50%), were operated on under general anaesthesia. The average weight of the pigs was 57 kg (SD 7.2 kg). The surgery was conducted under standard temperature conditions (23°C) in general anaesthesia using TKX (tiletamin-zolazepam + xylazin + ketamin). The laser-doppler probe holders were placed in the right and left groin. The island flaps based on arteria and vena epigastrica caudalis superficialis were elevated on both sides of the abdomen as shown on Fig 1. The vascular pedicle of the flap was exposed in the extent of 3 cm from its branching from the femoral artery and the 3-0 Polysorb suture was placed in the adventitia (Fig 2). Then, the flap was left resting for 20 minutes before the laser-Doppler probe (PeriFlux system 5000, small straight probe 407-1, Perimed, Jarfalla, Sweden) was attached and continuous recording of the perfusion signal began. After another 5 minutes of leaving the flap resting (this time point was assigned as t=0), the weight was attached to the suture and hanged on the block for 5 minutes. The weight of 160g placed in the direction of the vessel course produced consistent tension on the pedicle. In the treatment group A, the vessels were sprinkled with magnesium sulphate (Magnesium Sulphuricum Biotika 10%, Biotika, Slovenska Lupca, Slovak Republic), for the period of duration of the tension on the flap pedicle (t= +5 minutes) and continued for the next 2 minutes after the tension was released (t= +7 minutes). In the control group B, saline was applied in a similar way. Doppler signal recording continued for another 30 minutes.

The perfusion recording signals were exported from the control software package of the laser-Doppler flowmeter into ASCII format files. Graphic representations of blood flow were created from these files. Because the signals were corrupted by impulse noise, it made it impossible to clearly detect the important time points and signal amplitudes. Therefore, a Savitzky-Golay polynomial filter was employed to smooth the signals. Then, two important time periods “t_B” and “t_C” were extracted from the signals with the use of Matlab (The MathWorks, Inc) scripts. The time period “t_B” represented the period between t=+5[s] and the time point on the curve when perfusion began to rise after a period of poor perfusion secondary to the exposure of the pedicle to tension. This time was considered as the duration of the vasospasm. The time “t_C” represented the period between t=+5[s] and that point on the curve when the re-perfusion reached its maximal level (Fig 3).

The above-mentioned signal characteristics “t_B” and “t_C” were compared between the treatment group A and the control group B using the paired Student t-test. Values at the 5% probability level were considered statistically significant. Suitability of the paired t-test for the analysis was supported by the fact that the control and the treatment groups were on the same animals and by the high values of the Pearson’s correlation coefficients: Ro(tB_A,tB_B)=0.79 and Ro(tC_A,tC_B)=0,47. According to these findings, the two groups were supposed to be dependent samples.

RESULTS

Normality of distributions of the signal characteristics tB and tC was verified by visual inspection in the histograms of the measured values of tB and tC. Table 1 and Fig. 4, 5 show mean values and standard deviations of the variables tB and tC in the two analysed groups. The means of signal characteristics were compared with the use of the paired t-test and the results were as follows: the time tB, that represented duration of the vasospasm, was significantly shorter (P= 0.0236018 ) in the treatment group A than in the control group B and the time tC, that represented speed of flap re-perfusion, was not significantly shorter (P= 0.10087 ) in the treatment group A than in the control group B.

DISCUSSION

Several studies were dedicated to an investigation of the vasospasm on a porcine model^17-23. We used new experimental model that was influenced by previous studies on a rodent model (Vasospasm of the Flap Pedicle – The New Experimental Model on Rat). Our new experimental model was based on the axial pattern free flap. The vasospasm was repeatedly induced using exactly defined axial tension that was applied on the flap pedicle. The weight of the weight, that consistently and safely produced vasospasm, was determined on two pigs operated before this study by gradually adding the weights on the pulling thread. The weight of 160g consistently stimulated vasospasm in all subjects and vasospasm lasted long enough to allow the tested drug to show its effect. By removing adventitia in some extent, the tissue wrapped around vessels was insignificant and the force was applied straight on the vessel wall. Also, magnesium infiltrated the vascular wall easier.

The laser Doppler measure was chosen for flap perfusion monitoring, because of its accuracy and reliability. Also, monitoring of blood perfusion on the periphery of the flap encompassed any possible change in the blood flow inside the flap.^{24, 25} The laser Doppler in connection with computer as a recording device allows continuous recording of the signal amplitude. The signals were afterwards subjected to Savitzky –Golay filter to remove artificial peaks coming mostly from movements of the probe. Automated detection of the time values on the signal curves ensured maximum objectivity and accuracy in obtaining measured values.

The pathogenesis of vasospasm resulting from pulling the pedicle is still not clear. One of the possible explanations is direct myogenic response of the smooth vascular wall muscle. Stretching arterial smooth muscle to 1.2 times of its resting length results in maximal phosphorylation of myosin light chains and smooth muscle contraction.²⁶

Although the vasodilator properties of magnesium ions were well documented both in vitro^27-30 and in vivo^31-34, the exact mode of action of this drug on vasospasm is still not fully understood. The best explaining theory of mechanism of action is likely to be related to competitive inhibition between magnesium and calcium ions for binding sites on the myosin light chain kinase regulatory protein, Calmodulin. Calcium is unable to activate myosin light chain kinase when Mg²⁺ ion is bound to Calmodulin. This results in Mg²⁺ induced relaxation of smooth muscle fibrils due to conformational changes in the actomyosin ATP-ase, rendering it less active in a dose dependent manner.^35-37 Other explanation of magnesium ions action is inhibition of the release of excitatory amino acids and blockade of the N-methyl-D-aspartate-glutamate receptor.^{38, 39}

Altura et al. compared effect of Mg²⁺ ions with calcium blockers on the vessels. Data obtained from this study of different calcium channels (verapamil, nimodipine, nitredipine and nisoldipine) point at considerable heterogeneity of its active sites. Magnesium sulphate probably acted in all types of calcium channels of the vessels in different organs. Conversely, effect of calcium channels blockers is strongly organ dependent.⁴⁰

In vitro, Kimura et al. studied effect of magnesium on the vascular ring segments from human coronary arteries obtained by autopsy within 5 hours. Magnesium significantly inhibited the tonic contraction at concentrations of 1mM and 2mM, but increased the amplitude of periodic contraction. At concentration of 8mM reduced the amplitude of periodic contraction and tonic contraction of the rings.²⁹

Ram et al found that intravenous magnesium sulphate dilated the spastic basilar artery (provoked by the blood in the subarachnoid space) in the rat from 50% of the baseline diameter to 75% and topical application of magnesium sulphate dilated the spastic basilar artery to 150% of the baseline diameter.⁴¹

In clinical studies, Chia et al. proved positive effect on relieving cerebral vasospasm when plasma concentration was maintained at 1–1.5 mmol/l⁴² and magnesium was also efficient when the concentration was twice higher ⁴³. Van den Bergh et al.⁴⁴ and Wong et al.⁴⁵ confirmed these findings on cerebral vasospasm in pilot prospective randomized controlled studies. Magnesium therapy may be more effective if magnesium is administered as a preventive measure to protect against vasospasm rather than to treat a completely developed vasospasm.⁴⁶

We have been using perivascular (intra-adventitial) injection of magnesium sulphate empirically in microsurgical procedures from the 1990s – its effectiveness appears to be reliable despite the fact that no evidence has been provided yet.

Magnesium sulphate is a readily available, inexpensive substance that proved its efficacy in several experimental and clinical studies. This study confirmed our findings based on experimental study on a rodent model as well as our clinical observations. According to our knowledge, this is the first report that proved magnesium efficacy on relieving mechanically produced vasospasm of the flap pedicle on a porcine model.

CONCLUSION

Magnesium sulphate 10% shortened significantly the mechanically provoked vasospasm on superficial inferior epigastric flap in a porcine model. Further clinical studies are needed to prove the effect in humans.

Acknowledgement:

This study was supported by a grant from Internal Grant Agency of the Ministry of Health, Czech Republic, IGA - NR 8368-5. This work was also supported by the Ministry of Education, Youth and Sports of the Czech Republic (AdmireVet, CZ 1.05/2.1.00/01.0006 - ED 0006/01/01). Authors have neither conflict of interest nor funding support for publishing this paper.

Corresponding Author:

Libor Streit, M.D.

Department of Plastic and Aesthetic Surgery, St. Anne University Hospital

Berkova 34, 612 00 Brno, Czech Republic

E-mail: liborstreit@gmail.com