MINIATURE ELECTRODES FOR SMALL ANIMALS AND A METHOD OF USE

THEREOF

Field of the Invention

The present invention relates to miniature electrodes which are particularly useful for small animals such as rodents. More particularly, the invention relates to miniature bipolar electrodes which can be placed on organs of small animals with minimized trauma, and which are specifically useful for placing on moving organs, such as the epicardial surface of the heart, for example.

Background of the Invention

Rodents are extensively used in cardiac research partly due to their low cost and high applicability for genetic manipulations. However, techniques for studying in-vivo electrophysiology, specifically supraventricular, are limited due to the delicate structure of the atria and its small dimensions, which complicates the task of placing several electrodes in parallel. Transesophageal approaches usually do not enable high quality recordings from the rodent atria during programmed stimulation protocols due to the high stimulus intensity needed and the low resolution of the recorded signals.

Commercially available trans-venous electrodes designed specifically for rodent studies are expensive, but increase the quality of obtained signals. However, even with these devices the stimulus artifact usually obscure the atrial

signal during programmed stimulation protocols of the atria. For example, endocardial recordings are often carried out utilizing an electrophysiology catheter comprising a set of electrodes arranged on a distal section of the catheter (octapolar electrophysiology catheter of Scisense; ERP-800 of Millar Instruments) by introducing said catheter into the studied organ via a suitable body passage (e.g., blood vessels) . While these methods are minimally invasive properly positioning the electrodes over a desirable target tissue in the studied organ is a difficult task.

An epicardial approach for implantation of electrodes on the atria and ventricle was also published and is used by several laboratories (Berul et al., Circulation, 1996). This approach is demanding and highly invasive, necessitating large median thoracotomy and the use of sutures on the atria to place the electrodes. Due to the complexity of this methods, it is used by very few laboratories today, and the results are many times unsatisfactory. All the above described methods are currently applicable only for acute, short term studies in anesthetized rodents .

A temporary bipolar heart wire is described in US 5,871,528 wherein a proximal chest needle having a proximal pointed end, and a distal blunt end attached to at least two connectors by a weakened zone, is used for pacing, sensing or defibrillating by means of two electrodes disposed near the distal end of the wire, wherein the electrodes are formed of bare wire connected to respective electrical conductors which distal ends form the electrodes. Although it may be used for pacing, sensing, monitoring, or defibrillating, organs of humans or animals, it is not suitable for small animals, such as rodents.

The methods described above have not yet provided satisfactory solutions for obtaining high resolution electrophysiology readings from organs in small animals such as the atria, with minimal trauma and using relatively low costs means.

It is therefore an object of the present invention to provide a miniature electrode, and method of use thereof, for recording electrophysiology signals from organs in small animals with substantially high resolution.

It is another object of the present invention to provide a miniature electrode for recording electrophysiology signals from organs in small animals the manufacturing costs of which are relatively low.

It is a further object of the present invention to provide a miniature epicardial electrode, and method of use thereof, for recording electrophysiology signals from organs in small animals with minimal trauma, and which can be placed via a minimally invasive procedure.

It is yet another object of the present invention to provide a miniature electrode for recording electrophysiology signals from organs and for pacing organs in small animals, which may be used for long term cardiac (and specifically atrial) pacing\recording in awake, conscious and behaving small animals .

Other objects and advantages of the invention will become apparent as the description proceeds.

- A - Summary of the Invention

The present invention is directed to a miniature electrode device, and method of use thereof, which are particularly useful for electrophysiology research in small animals, such as rodents. The electrodes of the present invention comprise sharp hook-shaped attachment means capable of delivering electrical signals to, or from, a living tissue and allowing easily and precisely anchoring the electrodes to the wall of the organ to be studied, without the need of additional suturing. The sharp attachment means may be advantageously implemented by means of rigid electrically conducting pins. The attachment means are attached to electrically insulated flexible wires by means of suitable connecting means capable of providing the mechanical and electrical connectivity needed therebetween. The electrically conducting portions of the electrodes which are not electrically insulted are covered by an insulating material, preferably an insulating mold which covers a distal portion of the insulated wires, the connecting means, and most of the length of the conducting pins, leaving the hook-shaped attachment means uncovered.

The inventors of the present invention discovered that it is possible to obtain accurate and reliable electrophysiology measurement from living tissue and organs of small animals (e.g., rats and mice) by means of miniature electrodes comprising hook-shaped attachment means capable of delivering electrical signals to/from the studied tissue or organ, and capable of establishing relatively firm attachment by introducing said hook-shaped attachment means, or a portion thereof, into the studied tissue or organ. It was further found that the miniature electrodes of the present invention can be accurately placed at desirable locations on living

tissues and organs of small animals, and may be implanted in live animals for long term (up to 10-14 days) electrophysiological measurements .

The miniature electrode of the present invention is generally comprised of one or more flexible electrically conducting wires, one or more rigid hook-shaped attachment means, and connecting means capable of providing electrical and mechanical connection between said one or more electrically conducting wires and the one or more rigid hook-shaped attachment means, wherein said hook-shaped attachment means are capable of delivering electrical signals to/from the studied tissue or organ, and capable of establishing suitable attachment by introducing said hook-shaped attachment means, or a portion thereof, into the studied tissue or organ.

The rigid hook-shaped attachment means may be prepared from a piece of rigid electrically conducting wire or pin, having a length of about 5 to 10 mm, and diameter of about 0.05 to 0.2 mm, such as, for example, insect pins, one end of which is curved to form of a hook (having a diameter of about 0.2 to 0.5 mm) . An electrode head may be defined by the portion of the electrode comprising the one or more rigid hook-shaped attachment means and connecting means used for connecting the hook-shaped attachment means to the flexible electrically conducting wires. Advantageously, said electrode head is covered by electrically insulating mold, said mold preferably covers an end portion of the flexible electrically conducting wires, the connecting means, and a proximal portion of the hook-shaped attachment means, leaving the hooks of the rigid hook-shaped attachment means uncovered. The electrically insulating mold is applied such that an electrically insulating gap is obtained between electrically conducting

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- 6 - parts of the electrode (e.g., rigid hook-shaped attachment means), in order to prevent electrical short circuits.

In one aspect the present invention is directed to a miniature electrode device comprising one or more flexible electrically conducting wires, one or more rigid hook-shaped attachment means, and connecting means capable of providing electrical and mechanical connection between said one or more electrically conducting wires and the one or more rigid hook- shaped attachment means, wherein said rigid hook-shaped attachment means are capable of delivering electrical signals to/from the studied tissue or organ, and capable of establishing suitable attachment thereto by introducing said hook-shaped attachment means, or a portion thereof, into the studied tissue or organ.

Advantageously, the flexible electrically conducting wires comprise electrically insulating cover. The connection between the electrically conducting wires and the rigid hook-shaped attachment means may be also covered by an electrically insulating material (e.g., plastic, Teflon, Silicone Rubber, fluorocarbon or polyvinylchloride) .

The connecting means employed is preferably a type of crimpable metallic tube or a type electrically conducting glue.

Preferably, the flexible electrically conducting wires are made from silver, platinum, cooper or extra-flexible stranded silver-plated copper conductor.

The diameter of the flexible electrically conducting wires is preferably in the range of 50 to 200 micrometer.

The attachment means are preferably made from a narrow and rigid wire pieces or metallic pins having end portions curved in a shape of hooks, and having a diameter in the range of 0.05 to 0.3 mm, and sharp end portions (~12.5μm in diameter). Said attachment means are preferably made from stainless steel, platinum, or gold.

The electrode of the invention is preferably a bipolar electrode comprising two electrically conducting wires each of which connected to a respective rigid hook-shaped attachment means, wherein the electrically insulating material is a mold applied such that a gap (e.g., in range of 0.5 mm to 1.5 mm) is obtained between said rigid hook-shaped attachment means, and between the connecting means.

In another aspect the present invention is directed to a method for delivering, in-vivo or ex-vivo, electrical signals to/from a living tissue or organ of a small animal, comprising: forming an incision in said animal suitable for accessing said living tissue or organ, and in case of ex-vivo procedure the organ (e.g., heart) is hanged on a system were it is constantly perfused with physiologic solution and maintained at physiologic temperature, attaching to said living tissue or organ one or more hook-shaped electrodes, wherein said hook-shaped electrodes are capable of delivering electrical signals to/from said living tissue or organ and capable of being attached thereto by introducing their hooks thereinto; and applying or measuring electrical signals via said electrodes.

In case of in-vivo measurement, the method may further comprise suturing the incision and obtaining from, or

delivering to, the electrodes the electrical signals via flexible electrically conducting wires passed via the sutured incision, or wirelessly, by means of transceiver means connected to said electrodes.

Brief Description of the Drawings

The present invention is illustrated by way of example in the accompanying drawings, in which similar references consistently indicate similar elements and in which:

Fig. 1 schematically illustrates the structure of a miniature electrode according to a preferred embodiment of the invention;

Fig. 2 schematically illustrates placement of the miniature electrodes of the invention on an organ of a live animal; Figs. 3A to 3E schematically illustrate the steps of a preferred method for constructing the electrodes of the invention, wherein Fig. 3A demonstrates the preparation of the electrically conducting wires, Figs. 3B and 3C demonstrate attachment of electrically conducting pins to the wires, Fig. 3D demonstrates application of an electrically insulating cover over the exposed portion of the wires and over the attached pins, and Fig. 3E demonstrates exposing portions of the pins and forming hooks at their tips;

Fig. 4A provides a photographic presentation of a specific embodiment of the electrodes of the invention; Fig. 4B demonstrates utility of the bipolar electrodes of the invention in a hanging heart;

Figs. 5A to 5C demonstrate in-vivo use of the bipolar electrodes of the invention in ventilated anesthetized rats, wherein Fig. 5A demonstrates placement of the electrodes, Fig. 5B provides a photographic presentation of a ventilated anesthetized rat after placing the electrodes,

^■ _ g - and Fig. 5C shows graphs of signals measured via the electrodes;

Figs. 6A to 6D demonstrate implantation of the bipolar electrodes of the invention for long term atrial pacing/recordings, wherein Fig. 6A provides photographic presentation of the electrodes of the invention and of the interfacing connector, Figs. 6B and 6C provides photographic presentation of a live rodent after placing the electrodes of the invention and attaching the connector to its skin, and Fig. 6D shows graphs of signals measured via the electrodes;

Figs. 7A and 7B show a programmed stimulation protocol, and the consequent atrial electrophysiological measurements, in awake, freely moving rats; and

Figs. 8A to 8D show structure, application and measured results obtained with an embodiment of the invention employing electrically conducting glue, wherein Fig. 8A schematically illustrates the assembly of the electrodes, Fig. 8B provides photographic presentation of an electrode, Figs. 8C and 8D provides graphs of signals measured via the electrodes in a rat and a mice, respectively.

It should be noted that the embodiments exemplified in the figures are not intended to be to scale and are in diagrammatic form in order to facilitate ease of understanding and description.

Detailed Description of Preferred Embodiments

The present invention is directed to miniature electrodes designed for electrophysiology research in small animals such as mice, rats and Guinea-Pigs. The electrodes of the present

invention comprise hook-shaped attachment means which facilitates their placement on the outer wall of organs in small animals via small body incisions, with relative ease and with high precision. Moreover, the electrodes of the present invention are easy to manufacture and are relatively low-cost.

Fig. 1 schematically illustrates the structure of an electrode 10 according to a preferred embodiment of the invention. The tissue of the studied organ is electrically contacted to electrode 10 via a pair of rigid electrically conducting pins 13 the distal (leading) ends 13h of which are configured in the shape of a hook for allowing it to be easily and precisely anchored to the wall of the organ to be studied (20 illustrated in Fig. 2) . Electrically conducting pins 13 are electrically contacted to electrically insulated flexible wires 11 by connecting means 12 which provides both the mechanical and electrical connectivity needed therebetween. The electrical connection is achieved by removing a portion of the insulating coatings lib of insulated wires 11 for exposing a portion of their distal end conductors lie.

After achieving the connection between conducting pins 13 and the exposed conductors lie of insulated wires 11 by connecting means 12, the exposed electrically conducting portions of the electrode 10 (i.e., the portions which are not electrically insulted) are covered by an insulating mold 15. Said exposed electrically conducting portions are covered by the electrically insulating mold 15 such that a gap 17 (e.g., about 0.5 to 1.5 mm) is obtained between each of the assemblies consisting of the exposed conductors lie, connecting means 12 and electrically conducting pins 13, in order to prevent electrical short circuits therebetween. The

section of electrode 10 comprising electrically insulating cover 15 and conducting pins 13 define the electrode head 18.

Preferably, the insulating mold 15 covers electrode 10 starting from a distal portion of insulated wires 11, and continues toward the distal ends of conducting pins 13 by covering the exposed conductors lie of insulated wires 11, connecting means 12, and most of the length of conducting pins 13, while leaving the distal ends of conducting pins 13 comprising hooks 13h uncovered. Alternatively, electrically insulating mold may be applied over the entire length of pins 13 and a portion of their distal tips may be exposed by removing a respective distal portion of mold 15. In this case hooks 13h at the distal end portions of the exposed conductors lie are formed after removing the distal portion of mold 15.

Insulated wires 11 preferably comprise a small diameter flexible conductor lie having very good electrical conductivity. The diameter of conductor lie may generally be in the range of 50 to 200 micrometer, preferably about 125 micrometer, and its electrical conductivity may generally be greater than 10*10 ⁶ Siemens/m, preferably greater than 40*10 ⁶ Siemens/m, most preferably about 63*10 ⁶ Siemens/m, or greater. The insulating coating of insulated wires 11 may be made from any flexible electrically insulating material suitable for applying a substantially thin layer (e.g., about 75 micrometer) over the electrical conductors lie and providing electrical insulation, such as, but not limited to Isonel, Formvar, Teflon, silicone rubber, fluorocarbon, or polyvinylchloride . By way of example, insulated wires may be a type of silver wires coated by a Teflon layer, such as 786000 or 785500 Teflon® Insulated Silver Wires manufactured by A-M systems, INC. Of course, other types of insulated wires may be

used, for example, said wires may be made from platinum, cooper or extra-flexible stranded silver-plated copper conductor.

Attachment mean 12 are preferably made from an electrically conducting material capable of providing good mechanical and electrical connections. For example, attachment means 12 may be implemented by a type of electrically conducting glue, such as, but not limited to, Elecolit 325, or additionally or alternatively, by a crimpable electrically conducting tube. By way of example, an electrically conducting crimpable metallic tube may be used having an inner diameter generally in the range of 200 to 500 micrometer, preferably about 300 micrometer, and an outer diameter generally in the range of 300 to 650 micrometer, preferably about 350 micrometer. Such a metallic tube may be manufactured from a metallic material having good electrical conductivity, for example, a 26G stainless steel syringe needle may be used. The length of attachment means 12 may generally be in the range of 0.5 to 2 mm, preferably about 1 mm.

Electrically conducting pins 13 may be prepared from a relatively rigid and substantially thin metallic pin or wire. The length of conducting pins 13 may generally be in the range of 2 mm to 7 mm, preferably about 4 mm, and their diameter may generally be in the range of 0.05 to 0.3 mm, preferably about 0.1 mm. Conducting pins 13 are made from a good electrically conducting material, such as, but not limited to stainless steel or platinum preferably from stainless steel. Hooks 13h may be formed by means of high-precision tweezers or pliers, or any other suitable tool capable of curving the tips of conducting pins 13 into small rounded hooks 13h having a diameter generally in the range of 0.1 to 2 mm, preferably

about 0.5 mm. By way of example, conducting pins 13 may be implemented by a type of insect pins, such as, for example, Stainless steel minutien pins, catalog No. 26002-10, manufactured by Fine Science Tools (USA) , Inc.

The length of electrode head 18 may generally be in the range of 2 to 5 mm, preferably about 3 mm, and the diameter of electrically insulating mold 15 may generally be in the range of 1 to 3 mm, preferably about 2 mm.

As illustrated in the figures and exemplified herein, in a preferred embodiment of the invention electrode 10 is a bipolar electrode comprising a pair of electrically conducting pins 13 connected to a respective pair of electrically conducting wires 11. The distance between conducting pins 13 may generally be in the range of 0.5 to 2 mm, preferably about 1 mm. Of course, the electrode of the invention may be used for constructing other electrode arrangements comprising more electrodes (e.g., tri-polar, quad-polar, etc.) and having different geometrical shapes (e.g., planar, triangular, or having other polygonal cross-sectional shapes) .

Fig. 2 schematically illustrates connecting a set of bipolar electrodes 10 of the present invention to a studied organ 20. As shown in Fig. 2 due to their hooks 13h, electrodes 10 can be accurately and easily attached to the outer wall of the studied organ 20, thus allowing placing the same in a relatively simple procedure on an organ of a living animal with minimal trauma. The proximal (trailing) end of the insulated wires can be connected to almost any commercially available stimulator and/or amplifier 21 (e.g., ISO-Flex stimulator of A.M. P. I, or 1700 Differential AC Amplifier, of A-M systems, INC.) for generating stimulating signals to be

applied to said organ, and for measuring and recording signals received from said organ, via electrodes 10.

As will be exemplified hereinbelow electrodes 10 are suitable for long term monitoring applications in behaving rodents. In such applications electrodes 10 are placed on the studied organ 20 via a small incision and their proximal ends are then connected to a small connector that is attached to the animal skin, after which said incision is sutured. Following recovery of the animal, electrodes 10 can be connected to stimulating/amplifying means via the connector for applying and measuring signals to/from the studied organ.

Long term monitoring applications may be also implemented wirelessly. For example, the proximal ends of insulated wires 11 may be electrically connected to an implantable stimulating and signal processing unit (not shown) equipped with a suitable transducer for receiving external control signal to be submitted for generating and applying stimulating signals by said unit, and for transmitting respective indications concerning the signals measured via electrodes 10. Alternatively, said stimulating and signal processing unit may be attached externally to the body of the studied animal.

While the miniature hook electrode of the invention is exemplified and illustrated herein as a bipolar electrode, it should not be construed as limiting the scope of the present invention in any way. It should be understood that other electrode arrangements, such as unipolar, tri-polar, quad- polar, and so worth, may be easily constructed in much the same way within the scope of the present invention.

As will be shown in the following examples the electrodes of the present invention provides highly increased signal quality and reduced stimulus artifact. Other advantages of these electrodes in heart studies is that they can be placed both on the right and on the left atrium simultaneously, they are highly suitable for atrial electrophysiological measurements, Atrio Ventricular (AV) node conduction, and rapid atrial pacing, and that they can be used in a long term setup in which experiments can be carried out within periods of time ranging between several days to several weeks in awake behaving animals.

Example 1 - electrode construction

The following example describes a preferred method for constructing bipolar mini-hook electrodes of the invention. In this example, which is schematically illustrated in Figs. 3A to 3E, the electrodes were made from sharp stainless steel insect pins (100 μm in diameter, tip diameter 12.5 μm) which are attached physically and electrically to Teflon-coated silver wires. Said insect pins were coated with melting plastic up to their tips, which were then bent to form a double hook electrode.

A pair of Teflon-coated (lib) silver wires 11 (A-M systems, USA, Cat. No. 78600 or 785500) were turned one over the other in order to form a single-double stranded electrical wire (Fig.3A). The tips lie of the silver wires were exposed at their ends (2-3 mm) by removing a distal portion of the Teflon coating lib. Each tip lie was then attached to a miniature insect pin 13 (10 mm length, lOOμm diameter, 12.5 μm tip diameter FST, USA Cat. No. 26002-10) as follows: A) a 16G

L2008/000161

- 16 - stainless steel syringe was cut to create a short (about 1 nun) tube 12. B) The tube 12 was placed in a bench vice (not shown) , where the exposed tip lie of the silver wire 11 is inserted into the stainless steel tube 12 and the insect pin 13 is also inserted thereinto such that a distal portion thereof having a length of about half of the insect pin length (~5mm, Fig.3B) protrudes distally therefrom. The tube 12 was then squeezed by the bench vice to form an electrically conducting connection between the exposed tips lie of the silver wires and the insect pins 13 (Fig.3C). Following insect pin attachment to both silver wires, the pins were placed in parallel with an inter distance of ~lmm. The tip of the electrode including the stainless steel tubes and the insect pins were then coated with a delicate coating of hot melting plastic glue 15 (Fig. 3D) . After said plastic glue coating was solidified, the tip of said mold 15 was detached so that an uncovered length of the tips of the insect pins 13 (about 2-3 mm) were obtained. Using delicate forceps the tips were angled to form a pair of hooks 13h to be attached to the studied organ (Fig.3E) .

Example 2 - Hanging heart studies

In this example, shown in Figs. 4A and 4B, the electrodes 10 of the invention were easily attached to various places on heart 44 (Fig. 4B) . The connected electrodes 10 were able to move with the contracting tissue, thus enabling continuous pacing\recordings without disturbance to the mechanical activity of heart 44.

In this example Sprague-Dawley male rats (250-350 g) were anesthetized. The chest of said rats was opened and the heart

was excised and placed in cold Tyrode's solution. Canulation of the aorta was then preformed and thereafter the preparation was hanged on a conventional isolated heart setup and constantly perfused with heated (37°C) and oxygenated Tyrode's solution at a constant pressure of 100 mmHg, as shown in fig. 4B. Placement of electrodes for pacing\recording is routinely preformed in many laboratories using hanging heart setups. This task is usually complicated by the movement of the heart, which excludes the possibility of placing fixed electrodes as usually done with other preparations (e.g., brain slices). Heretofore a type of commercially available electrodes (e.g., MINI BALL JOINT HOLDER T32040, HUGO SACHS ELEKTRONIK - HARVARD APPARATUS GmbH) , specifically designed for hanging heart, were typically used for this purpose, where said electrodes are attached to the heart using flexible machinery. Achieving this task is substantially simplified and substantially less expensive by means of the bipolar mini-hook electrodes of the present invention.

Fig.4A shows a photograph of the tip of a bipolar mini-hook electrode 10 of the present invention. Electrodes 10 can be simply attached to any place on the atrial or ventricular epicardium of the hanging heart and can freely move with the myocardium. Therefore, electrodes 10 do not demand additional machinery for flexibility (Fig. 4B) . Since the tips of electrodes 10 are very delicate (e.g., about 12.5μm in diameter) the local damage to the tissue is minimal. In addition, since no additional machinery is needed to make the electrodes hanging, it is much easy to attach several bipolar mini-hook electrodes 10 in parallel to the same preparation.

Example 3 - Acute studies in-vivo

Using minimally invasive lateral thoracotomy two bipolar electrodes of the invention were placed on the right atrium (51 and 52 in Fig. 5A) , and a third bipolar electrodes on the right ventricle (53 in Fig. 5A) . Thereafter, high resolution recordings were performed, and various parameters were easily measured including: atrial refractoriness, AV refractoriness, atrial and AV conduction studies during continuous pacing including rapid pacing. It should be noted that it is very difficult to obtain such recordings in rats and mice with different modalities, and that placing these electrodes on the right and left atria in parallel is also feasible.

Use of bipolar mini-hook electrodes for in-vivo studies in ventilated anesthetized rats:

Rats (e.g., 48 in Fig. 5B) were anesthetized, intubated and ventilated with a rodent respirator (Inspira, Harvard apparatus) . Intubation was performed with a 14G catheter coated with silicone grease (excluding the tip) to prevent air leak and lung collapse upon thoracotomy. Positive-end expiratory pressure of 3 cm H ₂ O was applied continuously and increased to 5 cm H ₂ O upon thoracotomy. Maintenance of anesthesia was applied with 2.5 % isoflurane. Body temperature was continuously monitored with a rectal thermometer and was maintained at 37 ⁰ C by a heating pad. The animals were connected to an electrophysiological system (Nihon Kodhen) , and Surface ECG was monitored using cutaneous clips fixed on each limb. Right lateral thoracotomy was performed, and consecutive intercostals spaces 3 to 5 were gently exposed (Fig. 5B) . Pairs of miniature bipolar hook electrodes 10 were then inserted on the lateral aspect of the upper right atrium (51, URA) for pacing, and low right atrium (52, LRA) for

recording (Fig. 5A) . A third bipolar electrode was inserted on the right ventricle (53, RV) for assessment of the ventricular response. Electrophysiological signals were interfaced with a PC using a conventional A/D converter board (National Instruments) . A program written especially for the purpose of these experiments (created by the inventor with LabView 7.1, National Instruments) was used for electrical stimulation and signal analysis. Electrical stimulation consisted of square current pulses of 2 ms duration applied through an isolation unit (Iso-Flex, AMPI, Israel) . The frequency and duration of stimulation were controlled through the A/D board. Under these conditions, stimulus intensities as low as 0.05 mA can be obtained and clear differentiation between the stimulus artifact and the atrial signal could be easily obtained (Fig. 5C) .

Therefore, parameters such as Atrial effective refractory period (AERP) and Atrio-ventricular refractory period (AVERP) , minimal atrial 1:1 capture, and the Atrio-ventricular Wenckebach cycle length, which usually can not be recorded, are easily obtained. This holds true even if the obtained threshold intensity is higher (up to 0.5 mA) . Using this setup normal blood gases can be obtained up to 5 hours following thoracotomy, which indicates robustness of the experimental setup (table 1) .

Table 1

Example 4 - Instrumented conscious rodents

Heretofore, the prior art failed to provide techniques suitable for long term atrial pacing\recording in behaving rodents, a mode which is highly desirable. The miniature bipolar electrodes of the present invention can be easily placed and attached to the rodent myocardium including the atria, and can be used for various electrophysiological studies including implantation on the atria of behaving rats and mice.

Permanent implantation on the posterior part of the left atria enable long term (up to several days) atrial capture and recording through a small connector placed on the back of the behaving animal. Additional pairs of hook electrodes of the invention can be placed on different locations over the heart and used for recording of atrial\ventricular activity.

Implantation of bipolar mini-hook electrodes for long term atrial pacing\recordings :

For long term experiments in conscious rodents, 2-3 pairs of bipolar electrodes 10 of the invention as well as peripheral electrodes (for ECG measurements) were all connected to an 8 pin ^λ female' connector which is implanted in the back of the animal (40, Fig.6A). All portions of the instrument exposed to the animal tissue are covered with either plastic or Teflon. Sterilization was performed using electron beam radiation. Anesthesia was achieved using an intra-peritoneal injection of ketamine and xalazine. Rats were then intubated and ventilated in a "volume-cycled" mode with a rodent respirator (Harvard apparatus). The animal was warmed using a heating pad at 37°C. Under sterile conditions, one miniature bipolar hook electrode was attached to the posterior part of the left atrial tissue

through left lateral thoracotomy. This electrode was used for atrial pacing. A second bipolar electrode was attached to the right atrium using a tunnel under the skin and right minimal thoracotomy, for sensing of atrial activity. Using a third monopolar electrode on the back of the animal and one pole of the right atrial electrode allowed also recording ECG like activity, and follow the ventricular response. Following placement of the atrial electrodes in the correct position the back connector was inserted through a tunnel created in the animal's back and the chest incision was closed. Following chest closure, the animal was placed in prone position and the back connector was exposed and attached firmly to the back using a small sterile mesh, several sutures and super-glue application between the connector and the animal skin (fig.6B) .

Following the surgical procedure the animals were woken and allowed to recover for 3 days in a normal cage. During this period an analgesic (Dypirone) and an antibiotic (Ciprofloxacin) were added to the drinking water. Following the recovery period each animal was placed in a special recording chamber where the back connector was linked to an elastic suspension system, with a spiral spring, enabling the rat 60 to move freely in its cage (Fig. 6C), thereby permitting chronic pacing through the electrode as well as ECG and atrial recordings (Fig.6D). The rats received an adjustment period of 1 day to get used to the apparatus. Their spontaneous heart rate was recorded from the ECG electrode and attenolol (beta adrenergic blocker) was added to the drinking water (0.5-1 mg/ml) .

Following the recovery phase an initial burst pacing protocol was done to evaluate baseline AF induciblity, and then atrial

tachypacing, or sham treatment (pacing at a rate similar to the mean ventricular rate of the treatment animals) began at double threshold intensity. Repeated measurements were done twice daily for several days. While the system was highly suitable for studying long atrial tachypacing, other modalities such as: studying AV conduction, Sinus node function and drug effects can also be performed with the same apparatus. The system should also be useful for developing rodent models of pacemaker utilities such as biventricular pacing (mimicking cardiac resynchronization therapy - CRT) , pacemaker induced cardiomyopathy and tachycardia induced cardiomyopathy .

Example 5 - Atrial electrophysiological measurements in awake, freely moving rats.

Fig. 7A shows recordings of signals obtained from an animal in which one bipolar hook electrode of the invention was implanted on the left atrium for stimulation (LA bipolar stim. ) , and another such bipolar hook electrode was implanted on the right atrium for recording (RA bipolar rec). An additional reference electrode was implanted under the skin in the animal back, which allowed unipolar recordings against one pole of the RA hook electrode (RA unipolar rec). The procedure for electrode implantation is similar to that described in Example 4.

The plots in Fig. 7A, show signals of the RA bipolar and RA unipolar recordings during LA pacing at double threshold intensity. It should be noted that the RA atria signal is clearly discriminated from the stimulus artifact in both

recordings. Additionally, the RA unipolar recording also shows a high resolution signal of the ventricular activity.

The plots in Fig. 7B are of a bipolar RA recording obtained during a standard S1-S2 protocol preformed in order to determine the atrial effective refractory period at double threshold intensity. As seen in these plots, a successful atrial capture is seen in a time interval of Sl-S2=45ms, but it fails in a time interval of Sl-S2=42ms.

Example 6 - measurements carried in mice with electrodes constructed with electrically conducting glue.

Fig. 8A schematically illustrates an electrode 80 in which the exposed conductors lie of electrically insulated flexible wires 11 are attached to the rigid electrically conducting pins 13 by means of electrically conducting glue 84. The construction of electrode 80 is substantially similar to construction of electrode 10 described hereinabove, but it differs in that electrically conducting glue is employed for attaching pins 13 to conductors lie. As seen in Fig. 8B, electrode 80 is substantially small, in this implementation the gap between conducting pins 13 may be about 0.3 to 0.7 mm, the diameter of the electrically insulating cover 15 may generally be in the range of 0.5 to 1.5 mm, preferably about 1 mm, and the length of the electrode head 81 may generally be in the range 1.5 to 3 mm, preferably about 2 mm. The diameter of the attachment obtained by electrically conducting glue 84 may generally be in the range of 0.3 to 0.7 mm, preferably about 0.5 mm, and its length may generally be in the range 0.1 to 0.3 mm, preferably about 0.2 mm.

The setup in this example is substantially similar to that described with reference to fig 5A.

Figs. 8C and 8D provide graphs of signals measured via the electrodes 80 in-vivo in anesthetized rat and a mice, respectively. Three pairs of electrodes were implanted through a minimally invasive right lateral thoracoromy. One pair was placed on the high right atrium (HRA) for pacing. A second hook was placed on the low right atrium (LRA) for recording and a third hook was placed on the right ventricle (RV) for recording. It is noted the high quality electrical recordings were obtained during atrial pacing at the HRA electrode, with a very low stimulus artifact, and that high scale atrial and ventricular signals were obtained in the LRA and RV channels, respectively.

The above example shows that the bipolar electrodes of the invention are highly potent for the study of rodent electrophysiology in general, and supraventricular electrophysiology in particular. The cost of the electrodes of the invention is significantly lower than commercially available trans-venous electrodes and has conspicuous advantages over them for various modalities. The ability to implant electrodes in conscious rodents used for pacing the atrium of rats and mice for the first time may greatly facilitate rodent studies of atrial electrical characteristics and arrhythmias. The electrodes of the present invention can be easily attached to commercially available telemetric devices.

All of the abovementioned parameters are given by way of example only, and may be changed in accordance with the differing requirements of the various embodiments of the

present invention. Thus, the abovementioned parameters should not be construed as limiting the scope of the present invention in any way. In addition, it is to be appreciated that the different wires, connectors, and other members, described hereinabove may be constructed in different shapes (e.g. having cylindrical, square etc. form in plan view) and sizes differing from those exemplified hereinabove.

The above examples and description have of course been provided only for the purpose of illustration, and are not intended to limit the invention in any way. As will be appreciated by the skilled person, the invention can be carried out in a great variety of ways, employing more than one technique from those described above, all without exceeding the scope of the invention.