Trauma is the leading cause of death among US individuals younger than 44 years. Hemorrhagic shock accounts for 30–40 percent of traumatic mortality ^{1, 2} . Bleeding is also the most common preventable cause of death on battlefield ³ . Applications of tourniquets to compressible hemorrhages ^{4, 5, 6, 7, 8} caused a marked decrease in limb exsanguinations ^{3, 4, 9} . 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 ³ . 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 ¹⁰ . Though Combat GauzeTM 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 ^{9, 4, 3, 10} . A novel mechanical compressing device, the Combat Ready Clamp, was recently introduced into the US Army ^{3, 11} , But has not yet eat 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 ¹² , therefore major efforts are undertaken to develop technologies for this unmet need. In early 70 s, it was proved that thrombosis can be Inductive in a clamped blood vessel by minutes-long application of direct electric current ^{13, 14, 15} . 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 ^{16, 17} . More recently, constriction of blood vessels and thrombosis without thermal damage have been achieved with short ( μ s-ms) electric pulses ¹⁸ . However, these techniques have not been characterized In mammals, nor have they were evaluated for clinical use in

Recently we described significant decrease in blood loss from liver injury in rabbits treated by sub-millisecond electrical pulses 19 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 μ s per phase, amplitude of 250 V and repetition rate of 10 Hz caused, within seconds, a very pronounced local constriction of both femoral (Figure 1a,b) and mesenteric (Figure 1c,d) 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 Figure 2, as a function of stimulus amplitude and duration. Vasoconstriction increased with higher pulse amplitude and longer duration for both arteries and For all pulse durations tested, vessel diameter decreased with increasing pulse amplitude along a sigmoid curve, having a response threshold on the lower end, and reaching a minimum size of abou t 20–25% of the original diameter on the high end (Figure 2). 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 (Figure 3). 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 μ s/phase pulses of 250 V at 10 Hz repetition rate for 30 seconds. Mesenteric vessels before (c) and after (d) stimulation with 1 μ s 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 demonstrat that V ₅₀ and V ₂₅ 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 (Figure 4a) and veins (Figure 4b). 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 (Figure 4). 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 10 ³ Hz it was about 8 minutes.

A single site on the vessel was stimulated at V ₂₅ (80 V) with 1 μ s pulses for 2 minutes and allowed to recover without stimulation during 13 minutes. Repetition rate increased from 1 to 10 ⁴ Hz with increments of a factor of 10.

Response of blood vessels to continuous stimulation was measured during 10 minute intervals, with the frequency increment with each step, as shown in Figure 5. Again, the vessels reached the minimum size at each particular frequency within about a minute, and then at 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 (Figure 4) and continuous (Figure 5) stimulation, and the difference was more pronounced at higher repetition Rates. Maximum response to continuous stimulation (Figure 5) was smaller than to the transient stimulus regime (Figure 4), especially at higher repetition rates.

Blood vessels were stimulated at V ₂₅ (80 V) with 1 μ s pulses for 10-minute periods at each repetition rate. (MEAN +/− SE, n = 4).

Mesenteric blood vessels (Figure 6) 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 (Figure 2), and the difference increased with For example, with 1 μ s 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 μ s pulses of 150 V (corresponding to 75% constriction at 1 Hz repetition rate) at a repetition rate of 10 Hz for 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) (Figure 7). In all untreated animals, bleeding still continued after the 1 minute-long blood collection, and the animal died within Minutes of the time of the end of the measurements. when treated with a pulse amplitude of 30 V at 1 Hz (corresponding to 50% constriction threshold), there was no complete hemorrhage arrest, and therefore reduction in blood l Oss was less pronounced: (0.35 vs 1.05 ml, p = 0.005), as shown in Figure 7). Strong decrease in blood loss was also observed in the severed mesenteric arteries treated with 100 μ s pulses of 40 V at 1 Hz (corresponding To 75% constriction threshold), as shown in Figure 7.

After cutting, the femoral artery was stimulated for 30 seconds with 100 μ s 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 μ s pulses of 40 V (right white bar) at 1 Hz After 30 seconds, or not treated (red bar). In both vessels types, treatment caused decrease or even complete stoppage of bleeding after stimulation, while Northwest 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 μ s/phase, 10 Hz, 30 s) demonstrate 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 Supplementary Figure 1b). This estimate indicates that even the most humid regime of hemorrhage control did not engage thermal damage to the treated vessels. Modeling of resistive heating for reversible constriction (80 V, 1 μ s / 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 Figure 8. Figure 8a shows a cross-section of a femoral artery following complete vessel dissection and 30 seconds-long treatment with 100 μ s/phase pulses of 150 V at a The treatment caused complete occlusion of the vessel and cessation of bleeding within a few seconds. Upon euthanasia and tissue fixation the vessels dilated somewhat The the the the the the the the the The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The The 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 endotheli Um, media and adventitia of the blood vessel appear unaffected by the treatment.

(a) Femoral artery treated with 100 μ s 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. Blood in the control artery formed a detached clot during sample fixation.

Discussion

Microsecond electrical pulses can induce vasoconstriction within a few seconds, in both arteries and veins. Upon termination of stimulation, the blood vessels dilate back to their original size within a few minutes. This reversible vasoconstriction can be repeated, and it does not seem to involve The tissue damage. Upon permanent stimulation, a permanent blood clot may form, completely blocking the lumen of the blood vessel. Both the reversible vasoconstriction and irreversible clotting offer a powerful approach to hemorrhage control in non-compressible wounds. The extent of perfusion can be controlled Since varying the amplitude, pulse duration, and pulse repetition rate. Since the flow rate in a cylindrical pipe is proportional to the fourth power of the diameter (Poiseuille's equation ²⁰ ), even small constriction of the blood vessel should significantly reduce the flow.

Electrically induced vasoconstriction could result from several effects: stimulation of the sympathetic innervations of the blood vessels and direct stimulation of the smooth muscle ^{21, 22, 23, 24, 25} . We couldn't find reports of a similarly profound pharmacological vasoconstriction - down to </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt ^{; 24, 25} . Formation of the blood clot may result from localized vascular stasis or a response to injury of the vascular endothelium ^{18, 26, 27} .

Histological evaluation of the tissue up to 3.5 hours after vasoconstriction revealed no obvious damage. However, a longer follow-up is required to detect potential development of the inflammatory tissue response, apoptotic effects or other long-term manifestations of mild tissue damage. injury to smooth muscle in the blood vessels was observed one week following exposure to high electric field in rats 28, 29. injury to vascular endothelium can further enhance the blood clotting and thrombosis 30.

Conventional thermal coagulation of blood vessels typically requires tens of Watts of power delivered by electrocautery ³¹ or RF coagulator ^{31, 32, 33} . Such techniques cause significant tissue injury and are not efficient in coagulation of large vessels. New visualization techniques such as Ligasure can larger seals of arteries, but they require bulky power supply, good visualization of the vessel and access to the vessel from all sides for accurate positioning of the surgical probe, all of which prevent the use of this Technology in the field ³³ . In contrast, the low-power (few mW) electrical vasoconstriction helps reducing blood flow without thermal damage to the tissue, and may not require good visualization of the injured vessel for positioning of the tool. Is required for such stimulation, a small disposable device could be placed in the wounded area to reduce or stop local bleeding. Pulsatile muscle contraction in response to electrical stimulation could be minimized by using higher repetition rates.

In conclusion, electrical stimulation of vasculature by microsecond pulses can be used to control blood perfusion and reduce hemorrhage in non-compressible wounds. Temporary decrease in blood perfusion can be achieved in seconds using the reversible vasoconstriction regime, with vessels dilating back to their original size The modality could be used for non-damaging hemorrhage control in surgery and during trauma care. Permanent blockage of bleeding is achieved upon vasoconstriction followed by initiation of clotting. For practical use in trauma care and for treatment of the Battlefield injuries, a miniature device should be developed capable of delivering pulsed stimulation prior to arrival of the patient to the hospital. Due to low energy requirements a disposable battery-powered device can be just a few millimeters in width, so it can be inserted into The wound to stop traditional bleeding. alternative, a stimulator may remain outside th E body, and electric current can be delivered to the area of ​​interest via percutaneous penetrating needle electrodes, similar to tumor ablation by electroporation [ eg ³⁴ ] .

Methods