发布时间：2020-06-23 00:16 原文链接：医学研究：电刺激可帮助控制大鼠出血（三）

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 Supplementary Figure 1b). 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 Figure 8. Figure 8a 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 Figure 8b. 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.

Figure 8: Histology of the treated (a) and untreated (b) femoral artery following complete vessel dissection.

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 tissue damage. Upon stronger 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 by 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 equation²⁰), 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 almost complete obstruction. This suggests that electrical pulses can induce much stronger contraction of the smooth muscles than pharmacological agents. Interestingly, similar extent of electrically-induced vasoconstriction has been observed in-vitro, with modulation by pulse amplitude, duration and repetition rate^{23, 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. Long term 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 thrombosis³⁰.

Conventional thermal coagulation of blood vessels typically requires tens of Watts of power delivered by electrocautery³¹ or RF coagulator^{31, 32, 33}. Such techniques cause significant tissue injury and are not efficient in coagulation of large vessels. Typically, large vessels require mechanical ligation under direct visualization. Newer techniques such as Ligasure can thermally seal larger 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 field³³. 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. Since very low power 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 within minutes after termination of stimulation. This 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 local bleeding. Alternatively, a stimulator may remain outside the body, and electric current can be delivered to the area of interest via percutaneous penetrating needle electrodes, similarly to tumor ablation by electroporation [e.g.³⁴].

Methods

Animals

Wild-type Long Evans rats 55–67 days of age were used for all experiments (average weight 275 g). Animals were anesthetized with 75 mg/kg Ketamine HCl and 5 mg/kg Xylazine, with additional maintenance half-dose applied every 45 minutes. Buprenorphine (0.01 mg/kg) and Hartman's Lactated Ringer solution (114 mL/kg/24 hr 37°C) were applied sub-cutaneously before experiment for hydration and pain control. All animal care and experiments were carried out in accordance with guidelines for the humane care of animals and were approved by the Stanford Administrative Panel on Laboratory Animal Care.

Electrical stimulation setup

After anesthesia, dorsal side of the animal was shaved and a conductive gel (Signa Gel, Parker Laboratories, Fairfield, NJ, USA) was applied. Animal was placed in supine position on a stainless steel return electrode and internal body temperature was controlled at 37°C by a heating pad. Target vessel(s) were dissected from surrounding tissue and allowed to acclimate for 10 minutes before stimulation. A constant drip of 37°C Hartman's Lactated Ringer was kept on the exposed site throughout the experiment. Stimulation probe was fabricated from a 1 mL plastic syringe with 2 mm diameter opening, filled with normal saline (0.9% NaCl). A platinum electrode inside the syringe and return pad electrode were connected to a customized biphasic pulse generator and to an oscilloscope (Tektronix, Beaverton, OR, USA). Electric pulses were composed of two square phases of each polarity (positive followed by negative) separated by 10 μs. Pulse rising time was recorded shorter than 100 ns.

Measurements of the constriction threshold

Femoral vessels were stimulated at three sites beginning with the most distal one. Each site was stimulated for 10-seconds at 1 Hz repetition rate, and left untreated for 20 minutes for recovery. Another stimulation session was then applied to the same site. Mesenteric vessels were stimulated at the second bifurcation from the terminal arteries in intestines, and were treated only once. Images of the blood vessels before and after stimulation were taken with a digital camera (Sentech Inc., TC202USB-A), and the lumen diameters were measured using the ImageJ software.

Measurements of the frequency response

To assess frequency dependence of the vasoconstrictive response, femoral vessels were stimulated at the voltage levels corresponding to 25-percent constriction threshold as determined previously for 1 Hz. In one set of experiments the vessel was stimulated for 2-minutes at a certain frequency, and then allowed to recover without stimulation for 13-minutes. This was then followed by stimulation at higher frequency, with the same recovery period (Figure 4). In another set of experiments a continuous stimulation was applied at each frequency for 10 minutes without recovery periods between them. Stimulation probe was removed from the treatment site for a few seconds every minute to capture an image of the tissue. In each step of these experiments repetition rate was increased by a factor of 10 from 1 Hz to 10 kHz, and then back to 1 Hz.

Measurement of the bleeding rate from the injured femoral artery

Femoral artery was exposed and completely cut by surgical scissors. Animals in the treatment group received 100 μs electric pulses at 150 V and pulse repetition rate of 100 Hz for 30 seconds. In a control group electrode was similarly applied near the severed vessel, however no pulses were applied. In both groups hemorrhage volume was measured 1 minute after the vascular injury by soaking a cotton wool near the vessel and weighting the absorbed blood, as previously reported¹⁹. Statistical significance of the differences between the two groups was evaluated using Student t-test.

Histology

After stimulation the femoral and mesenteric veins and arteries were ligated with 6-0 nylon sutures, and the treated area was dyed with India ink for definitive identification in pathology. Tissue was dissected and fixed in 10% buffered formalin overnight at room temperature. Tissue was then dehydrated with a graded series of ethanols and fixed in paraffin, sectioned and stained with hematoxylin and eosin (H&E).

Computational modeling

The tissue temperature rise during electrical stimulation was estimated using an axio-symmetric 2-dimensional finite element computational model of the electric field and the resulting Joule heating (Comsol Multiphysics 4.1 software, COMSOL inc., USA). The geometry is illustrated in supplemental Figure S1, and the constants are listed in Table S1. In short, the model contained five key components: the muscle tissue, the blood vessel, the plastic pipette, the metal electrode, and the saline in the pipette and in a thin layer above the tissue.

The height of the disk electrode inside the pipette (approximately 2 mm) was calibrated with a saline-only model to match the impedance of 619 Ohms measured in a saline-filled petri dish. The tissue resistivity of 300 Ohm-cm was set in the full computational model to match the total impedance of 1.13 kOhm measured for the experimental setup. After solving the electrostatic field (Poisson) equation which defines distribution of the current density in space, the time-averaged heat source (Joule heating rate per unit volume) was calculated and then added to the Pennes bio-heat equation³⁵. To account for the blood flow in non-constricted vessel, a perfusion rate inside the blood vessel was set to 10.6 s⁻¹, corresponding to a flow rate of 1 mL/min through a 1 mm diameter blood vessel, where the heated length matches the pipette diameter. For treatment resulting in completely occluded vessels, this perfusion was removed.

The room temperature and pipette saline temperature was assumed to be 20°C, while the saline covering exposed muscle tissue was 37°C. An equilibration time of 100 s was used before calculating treatment-induced temperature rise to allow tissue temperature to approach steady state after contact with cooler pipette (see details in Supplementary Material).

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