醫(yī)學(xué)研究：電刺激可幫助控制大鼠出血

瀏覽次數(shù)：7941　發(fā)布日期：2013-7-25　 來源：《自然》中文版

Medical research: Electrical stimulation helps control bleeding in rats

本期Scientific Reports上發(fā)表的一項研究提出，用微秒脈沖對大鼠的靜脈和動脈進行電刺激，也許能幫助控制出血和減少不能通過壓迫止血的傷口中的失血。還需要進一步的研究來確定該方法對人類患者是否有效，但該方法對于在外傷中或手術(shù)過程中控制不能通過壓迫止血的出血也許會有用。

用止血帶止血在戰(zhàn)場上能幫助降低創(chuàng)傷死亡率，但該方法無法用于不能通過壓迫止血的出血，包括流入體腔（如腹腔）中的出血，也不能用于軀干與四肢或頸部接合部的傷口。上個世紀(jì)70年代，人們發(fā)現(xiàn)給一個被夾住的血管施加直流電幾分鐘時間可誘導(dǎo)產(chǎn)生血栓。然而，這樣所導(dǎo)致的熱損傷使得該技術(shù)無法用在臨床實踐中。

Yossi Mandel和來自Daniel Palanker實驗室的同事將微秒脈沖的電流施加到大鼠的大腿（腹股溝）區(qū)域和腹腔內(nèi)的靜脈和動脈上。這樣使得這些血管中在幾秒內(nèi)發(fā)生血管收縮，但幾分鐘內(nèi)血管又擴張回它們的本來大小。增強電流強度，血管被完全地、永久性地阻斷。大鼠股動脈和腹部動脈中的血流速度降低，出血被迅速止住，股動脈的失血量與未經(jīng)處理的大鼠相比減少7倍。作者在血管收縮后長達3.5小時未發(fā)現(xiàn)組織受到損傷，盡管還需要進行更長時間的跟蹤觀察來評估對組織是否會有任何潛在的長期影響。

Vasoconstriction by Electrical Stimulation: New Approach to Control of Non-Compressible Hemorrhage

Non-compressible hemorrhage is the most common preventable cause of death on battlefield and in civilian traumatic injuries. We report the use of microsecond pulses of electric current to induce rapid constriction in femoral and mesenteric arteries and veins in rats. Electrically-induced vasoconstriction could be induced in seconds while blood vessels dilated back to their original size within minutes after stimulation. At higher settings, a blood clotting formed, leading to complete and permanent occlusion of the vessels. The latter regime dramatically decreased the bleeding rate in the injured femoral and mesenteric arteries, with a complete hemorrhage arrest achieved within seconds. The average blood loss from the treated femoral artery during the first minute after injury was about 7 times less than that of a non-treated control. This new treatment modality offers a promising approach to non-damaging control of bleeding during surgery, and to efficient hemorrhage arrest in trauma patients.

Introduction

Trauma is the leading cause of death among US individuals younger than 44 years. Hemorrhagic shock accounts for 30–40 percent of traumatic mortality^, . Bleeding is also the most common preventable cause of death on battlefield. Applications of tourniquets to compressible hemorrhages^{, , , ,} caused a marked decrease in limb exsanguinations^{, ,} . As a result, according to the US army, hemorrhage not amenable to truncal tourniquets (also called non-compressible hemorrhage) is now the leading cause of preventable death. Part of the non-compressible hemorrhages occur due to bleeding into body cavities (such as the abdomen or chest), while others are caused by wounds in the junction between the trunk and the limbs or neck. The latter ones, called junctional hemorrhages, are recognized as a care gap, and those of the pelvic, buttock and groin area are of highest prevalence. Though Combat Gauze™ is endorsed by the US Army for bleeding care in areas not amenable to a tourniquet, it is often ineffective in junctional hemorrhages such as groin, gluteal, axilla, shoulder and others^{, , ,} . A novel mechanical compressing device, the Combat Ready Clamp, was recently introduced into the US Army^, , but has not yet been proven clinically. This device cannot be applied to wounds of the head, neck, abdomen and chest.

Effective prevention of blood loss in the pre-hospital arena offers the best opportunity to save soldiers with non-compressible injuries, therefore major efforts are undertaken to develop technologies for this unmet need. In early 70 s, it was demonstrated that thrombosis can be induced in a clamped blood vessel by minutes-long application of direct electric current^{, ,} . However, associated thermal damage precluded the use of this technology in clinical practice. Reduction in blood perfusion during electro-chemotherapy was also noted previously, and it was found to enhance the antitumor effect of the chemotherapy^, . More recently, constriction of blood vessels and thrombosis without thermal damage have been achieved with short (μs-ms) electric pulses. However, these techniques have not been characterized in mammals, nor have they been evaluated for clinical use in various bleeding scenarios.

Recently we described significant decrease in blood loss from liver injury in rabbits treated by sub-millisecond electrical pulses. The current study evaluates the effect of microsecond pulses on blood vessels in two areas non-amenable to truncal tourniquets: the groin area (femoral) and the abdominal cavity (mesenteric). We demonstrate significant vasoconstriction and decrease in blood loss following injury of these blood vessels. These results indicate a possibility of controlling non-compressible hemorrhage using non-thermal pulsed electrical stimulation.

Results

Constriction of femoral and mesenteric blood vessels

Stimulation current was applied to the exposed blood vessel via 2 mm diameter pipette electrode filled with saline, while a large pad return electrode was applied to skin on the back side on the animal via conductive gel. Biphasic (symmetric, anodic-first square) pulses of electric current with duration of 1 μs per phase, amplitude of 250 V and repetition rate of 10 Hz caused, within seconds, a very pronounced local constriction of both femoral () and mesenteric () arteries and veins. Extent of the constriction of femoral vessels in response to 10 seconds long stimulation at 1 Hz repetition rate is plotted in , as a function of stimulus amplitude and duration. Vasoconstriction increased with higher pulse amplitude and longer duration for both arteries and veins. For all pulse durations tested, vessels diameter decreased with increasing pulse amplitude along a sigmoid curve, having a response threshold on the lower end, and reaching a minimum size of about 20–25% of the original diameter on the high end (). Strength-duration dependence of the 25% and 50% constriction thresholds could be approximated by a power dependence t^−a, where a was approximately 0.3 for femoral arteries and veins (). For all pulse durations, lower voltage was required to reach similar constriction in arteries, compared to veins, although the difference decreased at longer durations.

Femoral vessels before (a) and after (b) stimulation with 1 μs/phase pulses of 250 V at 10 Hz repetition rate for 30 seconds. Mesenteric vessels before (c) and after (d) stimulation with 1 μs pulses of 80 V at 1 Hz for 30 seconds. Lumens of the femoral and mesenteric arteries and veins are indicated by the red and blue arrows, respectively.

Vessels sizes were measured after each stimulation session of 10 seconds in duration. Stimulation pulses were applied at 1 Hz. Vessels were allowed to recover for 20 minutes between the sessions.

Analytical fit demonstrated that V₅₀ and V₂₅ thresholds for both types of blood vessels scale with pulse duration as a power function of approximately ~t^−0.3.

Extent of vasoconstriction increased not only with the stimulus amplitude but also with the pulse repetition rate, for both the arteries () and veins (). In this set of measurements, stimulation was applied during 2 minutes, but the vessels constricted to the minimum diameter within about a minute. After the end of stimulation, the blood vessel slowly dilated back to its original width within about 10 minutes (). The recovery time to 90% of the original diameter increased with pulse repetition rate. For example, for 1 Hz it was about 4 minutes, while for 10³ Hz it was about 8 minutes.

A single site on the vessel was stimulated at V₂₅ (80 V) with 1 μs pulses for 2 minutes and allowed to recover without stimulation during 13 minutes. Repetition rate increased from 1 to 10⁴ Hz with increments of a factor of 10. 

Response of blood vessels to continuous stimulation was measured during 10 minute intervals, with the frequency increasing with each step, as shown in . Again, the vessels reached the minimum size at each particular frequency within about a minute, and then remained at approximately steady state, with the extent of constriction dependent on the repetition rate. Vasoconstriction was stronger in arteries than in veins for each pulse frequency with both transient () and continuous () stimulation, and the difference was more pronounced at higher repetition rates. Maximum response to continuous stimulation () was smaller than to the transient stimulation regime (), especially at higher repetition rates.

Blood vessels were stimulated at V₂₅ (80 V) with 1 μs pulses for 10-minute periods at each repetition rate. (MEAN +/− SE, n = 4).

Mesenteric blood vessels () had similar kind of response to that of the femoral arteries and veins. For the same pulse parameters, the extent of vasoconstriction in mesenteric arteries was higher than in femoral arteries (), and the difference increased with larger amplitudes. For example, with 1 μs pulses at 200 V mesenteric arteries constricted by 76%, compared to 49% reduction in femoral arteries. Mesenteric veins constricted more than the femoral veins at low amplitudes, while this ratio reversed at higher amplitudes.

Vessels were stimulated at repetition rate of 1 Hz during 10 seconds, with 20 minutes recovery between sessions. (MEAN +/− SE, n = 10).

Hemorrhage control during vascular injury

Complete cut of a femoral artery represents a model of traumatic injury leading to profound loss of blood by the animal. Applying 100 μs pulses of 150 V (corresponding to 75% constriction at 1 Hz repetition rate) at a repetition rate of 10 Hz for 30 seconds rapidly decreased the bleeding rate. In all 6 cases treated with this regime, a nearly complete hemorrhage arrest has been achieved within a few seconds. The average blood loss from the femoral artery measured during 30 seconds of treatment and 30 seconds after that was about 7 times less than that of a non-treated control (0.14 vs. 1.05 ml, p = 0.001) (). In all untreated animals, bleeding still continued after the 1 minute-long blood collection, and the animal died within minutes if bleeding was not mechanically stopped at the end of the measurements. When treated with pulse amplitude of 30 V at 1 Hz (corresponding to 50% constriction threshold), there was no complete hemorrhage arrest, and therefore reduction in blood loss was less pronounced: (0.35 vs 1.05 ml, p = 0.005), as shown in ). Strong decrease in blood loss was also observed in the severed mesenteric arteries treated with 100 μs pulses of 40 V at 1 Hz (corresponding to 75% constriction threshold), as shown in .

After cutting, the femoral artery was stimulated for 30 seconds with 100 μs pulses of 150 V at 10 Hz (white bar), or at 30 V and 1 Hz (pink bar). Blood was collected during stimulation and for an additional 30 seconds after stimulation. Control vessels were exposed and severed in a similar fashion, and the stimulation probe was placed above the vessel, but no stimulation was applied. Mesenteric vessels were treated with 100 μs pulses of 40 V (right white bar) at 1 Hz for 30 seconds, or not treated (red bar). In both vessels types, treatment caused decrease or even complete stoppage of bleeding after stimulation, while continuous bleeding was observed in the untreated arteries. Statistical significance of the differences between groups was evaluated using Student t-test: *p = 0.001,**p = 0.047 ***p = 0.005,****p < 0.001.

Finite element computational modeling of the resistive heating during the maximum exposure (150 V, 100 μs/phase, 10 Hz, 30 s) demonstrated that the peak temperature rise at the end of the treatment was between 2.3°C with full blood vessel perfusion and 2.9°C, with no blood perfusion (see ). This estimate indicates that even the most intense regime of hemorrhage control did not involve thermal damage to the treated vessels. Modeling of resistive heating for reversible constriction (80 V, 1 μs/phase, 10 Hz, 2 min) showed a temperature rise of less than 0.02°C, indicating that the mechanism of vasoconstriction is not thermal.

Histological findings

Histological sections of the treated and non-treated femoral arteries are shown in . shows a cross-section of a femoral artery following complete vessel dissection and 30 seconds-long treatment with 100 μs/phase pulses of 150 V at a repetition rate of 10 Hz. For comparison, an untreated control tissue from the other leg is shown in . Treatment caused complete occlusion of the vessel and cessation of bleeding within a few seconds. Upon euthanasia and tissue fixation the vessels dilated somewhat, compared to the most constricted state because of smooth muscle relaxation. In the middle of a circular lumen one can see an acute blood clot attached to the endothelium (solid arrow). The width of the blood clot illustrates the size of the blood vessel in its constricted state, prior to excision and fixation. The smooth muscle contraction is evident by circular appearance of the vessel and by dense folding of the internal elastic lamina (dashed arrow). The endothelium, media and adventitia of the blood vessel appear unaffected by the treatment.

(a) Femoral artery treated with 100 μs pulses of 150 V at 10 Hz for 30 seconds. Treatment caused complete occlusion of the vessel and termination of hemorrhage within a few seconds. Constricted lumen is filled with acute blood clot attached to the endothelium (solid arrow). Constriction is evident by round shape of the vessel and folding in the internal elastic lamina (dashed arrow). (b) Control femoral artery from the other side of the same animal was cut and left bleeding for 60 seconds. Free flowing blood in the control artery formed a detached clot during sample fixation.