Hemorrhage Control by Short Electrical Pulses

In Vivo Experiments

Guy Malki, Ofer Barnea and Yossi Mandel

Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv, Israel

Keywords: Hemorrhage Control, Electrical Pulses, Liver Injury.

Abstract: An internal hemorrhagic shock is one of the leading causes of death in the battlefield and other trauma

events. However the application of direct pressure, as in the treatment of an external hemorrhage, is not

possible. Most common techniques to achieve vasoconstriction are through heat; yet heating causes

irreversible destruction of organ tissues. Therefore, there is a need for a non-thermal based technology for

hemorrhage control. The current research describes, for the first, an attempt to reduce the amount of

bleeding in animal model liver injuries by using electrical pulses treatment (EPT). In the experiments, which

were performed on 28 rats and 14 rabbits, a short (25µs and 50µs) EPT was applied to the treatment groups

and the amount of bleeding was compared to the non-treatment (NT) groups. A reduction of 60%, 36% and

44% in blood volume, was found in the 25µs-rats, 50µs-rats and 25µs-rabbits EPT groups, respectively

(P<0.001). Also, it was found that the hemorrhage control was not caused by the mechanical pressure

applied by the electrodes, and there was no evidence for thermal coagulation. Further research is needed to

fully expose the potential of this treatment and the modality for hemorrhage control in civilian and military

settings.

1 INTRODUCTION

Hemorrhage shock is one of the leading causes of

death in the battlefield and other trauma events.

Most battlefield hemorrhages are compressible, e.g.

they can be controlled by a tourniquet or other

means of direct pressure application. A recent

survey made by the US army demonstrated the

effect of early tourniquet application in increasing

survival rates while causing minimal damage (Kragh

et al., 2011).

However, bleeding occurring in internal cavities

(such as the chest or the retroperitoneal space) or in

solid organs (e.g. liver, spleen and kidneys) is

considered non-compressible and the application of

direct external pressure is not possible. Hemorrhage

control from solid organs is challenging even in the

setting of an operation theatre, because of their rich

vasculature and lack of supportive connective

tissues.

In order to cope with this important need, there

are several techniques that being researched for

hemorrhage control in solid organs, of which the

main ones are by mechanical pressure and thermal

coagulation. However, each of these technologies

has its drawbacks and currently none of them have

evolved into clinical devices. For example, High

Intensity Focused Ultrasound (HIFU) (Burgess et

al., 2007); (Vaezy et al., 1997), induces a rapid

temperature increase in tissue, and cavitation

formation, both leading to thrombosis and platelet

activation. This technique has some adverse

reactions such as an irreversible destruction of the

liver and blood vessels, and overheating.

Nevertheless, these techniques, as well as others, are

still under evaluation and were not proved to give

full answer the clinical need in the battlefield or in

the surgical theater. Thus, there is a need for a non-

thermal based technology which will cause

vasoconstriction, thrombosis and hemorrhage

control.

Thrombosis of a clamped blood vessel was

demonstrated by several authors by using a direct

current application device (Guarini, 1971);

(Hladovec, 1975). However, applying this technique

for clinical use in hemorrhage control is not practical

because of the expected injury to the tissue. The

effect of short electrical pulses on blood vessel

constriction and thrombosis was reported in several

papers (Gehl et al., 2002); (Matsushima et al., 1994);

103

Malki G., Barnea O. and Mandel Y..

Hemorrhage Control by Short Electrical Pulses - In Vivo Experiments.

DOI: 10.5220/0004190001030107

In Proceedings of the International Conference on Biomedical Electronics and Devices (BIODEVICES-2013), pages 103-107

ISBN: 978-989-8565-34-1

 2013 SCITEPRESS (Science and Technology Publications, Lda.)

(Sersa et al., 1999); (Yu-ling et al., 1997), which

studied electrochemical therapy for tumors. These

papers demonstrated a significant temporary

reduction in blood flow after pulsing.

The specific characterization of various pulse

parameters on blood vessels constriction and

thrombosis was recently described by Palanker et al.,

(2008), which reported that the vasoconstriction

effect appeared shortly after 10 seconds of electrical

stimulation while thrombosis was achieved at 3

minutes. A pulse rate of 0.1Hz was sufficient for

maintaining vasoconstriction, yet there were

evidence of inflammation and necrosis. The authors

reported that the effect is probably non-thermal as

the temperature rise during the treatment did not

exceed 0.01

C. Nevertheless, the exact mechanism

for vessel constriction and thrombosis is still

unclear, thus a comprehensive study of these

mechanisms is necessary.

The potential application of the electrical pulsing

effect on blood vessels is studied by our group. Our

long term goal is to develop a portable device to

control internal non-compressible hemorrhage from

solid organs. The current research describes, for the

first time in vivo experiments, an attempt to reduce

the amount of bleeding caused in rat and rabbit liver

injury.

2 METHODS

In vivo experiments were performed on livers of 28

adult Sprague-Dawley rats and 14 New Zealand

rabbits. The experiment protocol was approved by

the Animal Rights Council of the Israel Ministry of

Health and conformed to guidelines for the humane

care of animals. Animals were supplied by Harlan

Laboratories Ltd., Jerusalem, at the age of 3 months.

Average animal weight is depicted in Table 1.

2.1 RAT Experiments

The animals were anesthetized with an IM injection

of Ketamine HCI (0.19ml/100 gram) and Xylazine

(0.03ml/100 gram) solution. The surgical operation

started 20 minutes after anesthetization to get an

initially uniform point for all animals, and until that

time the animal was weighed and placed on a

heating blanket to maintain its body core

temperature. Additional anesthetics were given

approximately every 20 minutes via titration. A

midline abdominal incision was performed and the

liver was gently exposed. The median lobe of the

liver was resected 13 mm from the lobe edge and the

removed part was weighed using portable scales

(Ohaus Company, model N2B110).

Following the liver injury, the animals were

divided into four groups. In the control group no

treatment (NT) was given after the liver injury.

Electrical pulses treatment (EPT) was performed to

2 groups, using a protocol which includes 100

electrical pulses of 500V at a pulse repetition of

1Hz, and pulse duration of 25µs and 50µs, for the

EPT25 and EPT50 groups, respectively. In addition,

in order to inspect the possibility of bleeding

decrease due to mechanical pressure exerted on the

liver lobe, a mechanical treatment (MT) group had

been defined, so that the electrodes were placed on

the median lobe for 200 seconds, as similar to the

EPT groups, however, no pulses were delivered.

For all the groups, except the NT, the rat’s

injured medial liver lobe was placed between two

customized copper electrodes, which were attached

to a commercial caliper (Figure 1a). The distance

between the 2 parallel slabs was adjustable, and

determined for each animal by its liver thickness

(mean electrode distance was 3.98±0.56 mm). A

series of electrical pulses were generated by a square

wave electroporation system (ECM 830, Harvard

Apparatus), which was operated in the mono-phasic

mode.

Following these interventions, the abdomen was

closed using continuous sutures and the rats were

maintained on heating blanket for 1 hour without

any further treatment. Total blood loss was

measured 60 minutes after liver injury, by soaking a

cotton wool in the peritoneal cavity; the same

method reported by previous authors (Hildreth et al.,

1996); (Holcomb et al., 1999); (Matsuoka and

Wisner, 1996). Blood loss for each animal was

normalized by its body weight. All surgical

interventions and measurements were performed by

the same investigators (GM, YM), to avoid variance

in the procedure, which could affect the results.

The liver was removed immediately after

euthanasia and fixed into formaldehyde 10%.

Histological slices were processed for H&E staining

in paraffin sections and then cut perpendicular to

liver edge in order to demonstrate the transition

between treated and untreated zones.

2.2 Rabbit Experiments

Animals were anesthetized with an intra-muscular

injection of Ketamine HCI (50 mg/kg) and Xylazine

(3.5 mg/kg) solution followed by maintenance

dosage, and 20 minutes following the administration

of anesthetic drugs, a midline abdominal incision

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Table 1: Animal weight and excised liver weight (normalized to animal weight) in all groups.

Rat Rabbit

Group NT MT EPT50 EPT25 EPT25 NT

11 4 8 5 7 7

Animal weight [gr]

398.66±20.14 408.3±49.5 398±32.9 392.3±33.6 3196±448 3106±293

Normalized Excised

Liver Weight [%]

0.26±0.07 0.25±0.06 0.22±0.05 0.21±0.05 - -

was performed and the liver was gently exposed. In

this experiment set, different liver injury was

performed: 2 cuts of 5mm deep and 3cm long in

each front liver lobe (total 6 cuts in 3 lobes).

Following liver injury, rabbits were divided into

two groups. EPT25 group received 200 pulses

(500V, 1Hz) that were given with pulse duration of

25µs and NT group received no treatment. In the

EPT25 group, the rabbit liver was treated with

customized electrodes comprised of two copper

plates of 4mm wide and 29mm long, and spaced

apart by 7.2mm (Fig 1b). Following each liver cut,

the electrodes were positioned on the liver surface

while the cut is in equal distances between the

electrodes and a series of electrical pulses were

given, similarly to the treatment described for rat

liver injury. In addition, thermal images have been

taken using a thermal camera (model A40, FLIR)

before and immediately after the treatment, in order

to distinguish if there is temperature increase during

the electrical treatment.

Following these interventions, rabbit’s abdomen

was closed using continuous sutures and the animals

were maintained on heating blanket for 1 hour

without any further treatment. Total blood loss was

measured 60 minutes after liver injury in the same

method as described for rats. All surgical

intervention and measurements for all experiments

were performed by the same investigator (GM).

3 RESULTS

Average animal weight and excised liver weight in

rats (normalized to the animal weight) are reported

in table 1. Both parameters were not significantly

different between all animal groups (p>0.1). These

results indicate that the injury protocol was pretty

much the same, and therefore could not affect the

blood loss results.

Blood loss weight can be observed in Figure 2,

as a box plot chart of normalized bleeding weight for

all animal groups. On each box, the black circle is

the mean value, the central line is the median, the

edges of the box are the 25

and 75

percentiles, and

the lines outside the box are the most extreme data

points (minimum and maximum).

In contrast to the normalized excised liver

weight, the blood loss amount in the EPT50 and

EPT25 rat groups was significantly reduced by 36%

and 60%, respectively, as compared to the NT group

(p<0.001 for both groups). Blood loss in the EPT25

group was significantly lower than in the EPT50

group (p=0.025), suggesting that the pulse duration

can affect the molecular processes of the electrical

treatment (which are not known yet) in becoming

faster and/or more efficient. Blood loss in the MT

group did not differ significantly from the NT group

(p=0.43), and this finding is probably indicates that

the mechanical pressure is not the reason for the

reduced bleeding from the injured liver.

Similar results were found for the rabbits (Fig 2),

where blood loss in the electric pulse treatment

group was smaller by more than 44% as compared to

the NT group (p=0.004).

4 DISCUSSION

The results, which were achieved by in-vivo animal

experiments, demonstrated that short electrical

pulses of 25µsec and 50µsec decreased the amount

of hemorrhage from a rat liver injury by 60% and

36%, respectively, and a rabbit liver injury by 44%.

Apparently, the effect was not caused by the

mechanical pressure applied by the electrodes per-

se, but by the electrical field applied on the tissue,

because there was no significant different in the

measured bleeding amount between the NT group

and the MT group.

The distinction between the treatment effective

between the rats and the rabbits can be explained by

the difference in the electrode configuration. In the

rabbits’ case the treatment was on the surface area,

so the electric field was not enough deep. On the

other hand in the rats’ case the electrodes were in the

two sides of the wound, so the electric field was

stronger in the injury site, and increase the treatment

effective.

Another potential cause for hemorrhage

reduction could be a thermal coagulation caused by

the increased temperature rise in response to pulse

HemorrhageControlbyShortElectricalPulses-InVivoExperiments

105

Figure 1: Experimental setup of rat (a) and rabbit (b) liver. Rat liver was treated with two parallel plate electrodes mounted

on a hand caliper adjusting for liver thickness. Rabbit liver injury was treated by fix parallel plates electrodes positioned at

two sides of the wound.

Figure 2: Box plot of normalized bleeding weight in all animal groups (rats and rabbits). Control groups were not treated,

EPT50 and EPT 25 were treated by 200 pulses of 50 and 25 µs, respectively, in a repetition rate of 1 Hz. Unpaired t-test

results for various comparisons are as follows: (*) p<0.001, (**) p=0.43, (***) p=0.025, (****) p=0.004.

treatment. However, thermo-coagulation is usually

expected at temperatures of above 60-70

C (Graham

et al., 1998); (Matsuoka et al., 2004), and were

probably not achieved in these experiments, even at

the longer pulse duration [According to a parallel

theoretical study in our group, which investigated

the shape of the electric field and the heating that

accompanying to the electrical treatment in different

configurations of electrodes, through mathematical

models and computer simulations in COMSOL and

MATLAB].

Further, there was no evidence for thermo-

coagulation and temperature rise in the histological

sections and the thermal images, respectively.

Interestingly, we found that 25µsec pulses were

significantly more effective in reducing hemorrhage

volume compared to 50µsec pulses. One possible

explanation may be related to a local increase in

liver perfusion in response to the relative

temperature rise in the case of 50µsec pulses, as

reported in previous studies (Precup et al., 2010). It

could be hypothesized that such a local increase in

perfusion could cause a relative increase in blood

loss, partially reducing the effect of treatment.

We hypothesize that the hemorrhage control

observed in this study is associated to endothelial

layer damage, leading to irreversible vessel

constriction and forming a thrombus. A similar

effect was reported in other electrochemical therapy

studies (Gehl et al., 2002). Ramirez et al., (1998)

reported that electrical pulses of 850 V/cm and 100

microseconds long, caused a decrease in blood

perfusion to the spleen and mesenteric arteries, and

also reported that electrical pulsing of the liver

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caused a decrease in perfusion, as was demonstrated

by a color test. Sersa et al., (2008) found that 3

minutes following tumor electric pulsing, blood flow

decreased by about 80 percent, and histological

evaluation of the endothelial cells showed that they

were rounded and swollen causing narrowing of the

blood vessels lumen.

Our study has several limitations to be

considered. First, blood pressure and pulse were not

measured or controlled during the experiments. This

could theoretically increase variance in the amount

of bleeding. Second, in this preliminary study we did

not address the effect of treatment in the case of

traumatic coagulopathy, which is expected in cases

of severe liver trauma. This issue calls for future

research. Other issues to be studied in larger animals

are the design of the electrodes in order to optimize

electric field geometry, optimize pulse parameters to

achieve finer results, better control of tissue

temperature, and the possible use of changes in the

electrical properties of the tissue for measuring

treatment effect.

In conclusion, in this preliminary research we

demonstrate that short electric pulses can

significantly reduce the amount of bleeding from

injured liver in a rat model. The effect is probably

non thermal and possibly related to the effect on

blood vessels’ endothelial layer. Further research is

needed in order to fully expose the potential if this

treatment modality for hemorrhage control in

civilian and military settings.

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