Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
  • A61N1/00—Electrotherapy; Circuits therefor
  • A61N1/18—Applying electric currents by contact electrodes
  • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
  • A61N1/326—Applying electric currents by contact electrodes alternating or intermittent currents for promoting growth of cells, e.g. bone cells

Definitions

• the present invention provides a novel stimulatory device for the controlled production of angiogenic growth factors. More specifically, the present invention provides a subthreshold pulse generator for the local production of vascular endothelial growth factor.
• Coronary artery disease results from arteriosclerosis of blood vessels serving the heart.
• Arteriosclerosis is a hardening and narrowing of the arteries.
• CAD Coronary artery disease
• the ischemic area The ischemic area of the heart, because it does not get adequate blood flow, is starved of oxygen and nutrients. This blockage, if not treated quickly, can lead to severe tissue damage.
• surgical procedures are used to graft new blood vessels to the ischemic area to improve circulation.
• angioplasty or stenting of the blocked blood vessel is done to reopen or maintain blood flow.
• bypassing or reopening of the arteries is often not possible because of limitations of present methodologies and the risk to the patient from surgical intervention. Damage from ischemia from insufficient blood circulation can also occur in blood vessels peripheral to the heart.
• Peripheral arterial occlusive disease (PAOD) caused by arteriosclerosis or by formation of vascular blood clots from diseases such as diabetes, often leads to loss of external limbs.
• Angiogenic factors are known to directly participate in the formation of new blood vessels.
• Local administration of recombinant angiogenic growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) can salvage ischemic areas of myocardial and skeletal muscle tissue in animal models.
• bFGF basic fibroblast growth factor
• VEGF vascular endothelial growth factor
• a number of approaches have been developed to deliver these factors to ischemic areas in hope of developing new blood vessels, including direct injection, electroporation, and delivery using retro viral vectors.
• angiogenic growth factors has many problems associated with it, most notably problems with effective delivery of the factors into the cells. Electroporation is a possible method of delivery of genetic materials encoding angiogenic factors; however, the transfection efficiency is still very low and the high-energy pulses directed to the tissue often kill many healthy cells. Alternatively, others have sought to develop viral based gene delivery systems to directly produce angiogenic factors in vivo; however, this approach requires considerably more development before it is considered to be a safe and effective therapy. Although extensive research continues in the areas of gene delivery, very little has been reported on methods to control and regulate gene expression in vivo. The inability to effectively deliver the agent to the target tissue, therefore, is one of the major limitations of the use of such agents. During delivery of the angiogenic factors the effectiveness is often destroyed or lost.
• the present invention addresses a number of problems existing in the prior art with respect to controlled local delivery of angiogenic factors.
• Various embodiments of the present invention provide solutions and to one or more of the problems existing in the prior art with respect to delivery of angiogenic factors.
• the present invention provides a novel electrical pulse generator for angiogenesis and production angiogenic growth factors.
• the present invention provides an electrical pulse generator for providing subthreshold pulses.
• the present device can be adapted to a range of subthreshold pulses by modulating the time, frequency, and delivery of a given stimulus.
• the present generator allows the use of a constant voltage, regardless of the distance between electrodes by allowing a variable field density.
• the present generator allows for control over the amplitude of the voltage and for charge balancing of the delivered and recovered charged pulse.
• the subthreshold pulse generator can be used externally, but preferably is designed and configured to be implantable.
• the subthreshold pulse generator includes a power supply and a control mechanism interconnected with the power supply.
• the pulse generator can be used with a plurality of electrodes in electrical communication with the power supply.
• the present generator is also capable of checking the lead continuity at a predesignated time.
• the invention also provides a subthreshold pulse generator for a patient in need thereof.
• the invention includes a method for reducing or repairing tissue injury or disease by providing a means for regulating angiogenic growth factor production.
• the subthreshold stimulation provided is sufficient to stimulate angiogenesis in the targeted body tissue.
• the present invention provides a novel method of pacing that is capable of stimulating cells or tissues for the controlled expression of angiogenic factors.
• Figure 1 Subthreshold Stimulation of Heart Tissues For Production of VEGF.
• Figure 1 is an overview of one mode of operation for subthreshold stimulation of cardiac tissue.
• FIG. 1 Simplified Schematic of The Output Circuit for Subthreshold Stimulation
• Figure 2 illustrates the schematic of the output circuitry of a subthreshold stimulation device for a pulse generator.
• Figure 3 Equivalent Circuit of the Subthreshold Stimulation During the Output Stage
• Figure 3 illustrates the schematic of the output circuitry of a subthreshold device for a pulse generator during the output stage.
• Figure 4 illustrate a pacing scheme for providing a series of subthreshold stimulations.
• Figure 5 Pulse Generator for Subthreshold Stimulation
• Figure 5 shows a block diagram of a circuit for pulse generator capable of delivering electrical stimulation to the target tissue cells.
• FIG. 6 Schematic of The Output Circuit for Subthreshold Stimulation
• Figure 6 illustrates the schematic of the output circuitry of a subthreshold stimulation device for a subthreshold pulse generator.
• FIG. 7 VEGF Production in Stimulated and Unstimulated Tissue.
• Figure 7 shows a western blot of VEGF protein in stimulated and unstimulated vascular tissue.
• Angiogenic factors are a group of substances that promote angiogenesis in a tissue.
• VEGF vascular endothelial growth factor
• FGF fibroblast growth factor
• mature protein or "mature polypeptide” as used herein refers to the form(s) of the protein produced by expression in a mammalian cell. It is generally hypothesized that, once export of a growing protein chain across the rough endoplasmic reticulum has been initiated, proteins secreted by mammalian cells have a signal sequence which is cleaved from the complete polypeptide to produce a "mature" form of the protein. Often, cleavage of a secreted protein is not uniform and may result in more than one species of mature protein. The cleavage site of a secreted protein is determined by the primary amino acid sequence of the complete protein and generally cannot be predicted with complete accuracy. However, cleavage sites for a secreted protein may be determined experimentally by amino-terminal sequencing of the one or more species of mature proteins found within a purified preparation of the protein.
• “Operably coupled” refers to the transference of an electrical stimulus by a subthreshold pulse generator to a tissue.
• a subthreshold pulse generator operably coupled with tissue or cells refers to a configuration where an electrical stimulus is delivered to the tissue or cells to cause an increase in available angiogenic factors. Usually the stimulus is delivered from the subthreshold pulse generator through leads to electrodes attached to the tissue
• treating refers to curative therapy, prophylactic therapy, and preventive therapy.
• An example of “preventive therapy” is the prevention or lessening of a targeted disease or related condition thereto.
• subthreshold stimulation can be used prophylactically to promote angiogenesis as a preventive effort to avoid the occurrence of a myocardial infarction.
• Those in need of treatment include those already with the disease or condition as well as those prone to having the disease or condition to be prevented.
• treating also describe the management and care of a patient for the purpose of combating a disease or related condition, and includes the administration of at least one subthreshold electrical pulse to an ischemic area to improve blood flow to the tissue.
• Chronic administration refers to administration of an electrical stimulus in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
• Ischemia is defined as an insufficient supply of blood to a specific organ or tissue. A consequence of decreased blood supply is an inadequate supply of oxygen and/or nutrients to the organ or tissue. Prolonged ischemia may result in injury to the affected organ or tissue. "Anoxia” refers to a virtually complete absence of oxygen in the organ or tissue, which, if prolonged, may result in death of the organ or tissue.
• Ischemic injury refers to cellular and/or molecular damage to an organ or tissue as a result of a period of ischemia and/or ischemia followed by reperfusion.
• Hydrophilic condition is defined as a condition under which a particular organ or tissue receives an inadequate supply of oxygen.
• Anoxic condition refers to a condition under which the supply of oxygen to a particular organ or tissue is cut off.
• Reperfusion refers to the resumption of blood flow in a tissue following a period of ischemia.
• patient refers to any mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as cattle (e.g., cows), horses, dogs, sheep, pigs, rabbits, goats, cats, and non-domesticated animals such as mice and rats.
• the mammal is a human, dog, rabbit, or mouse.
• a "therapeutically effective amount” as referred to herein is the minimal amount of subthreshold stimulation that is necessary to impart a therapeutic benefit or a desired biological effect to a patient.
• a "therapeutically effective amount” for a patient suffering from ischemia is such an amount which induces, ameliorates, or otherwise causes an improvement in the amount of angiogenic factors available or otherwise improve circulation in the tissue.
• a "therapeutically effective stimulus” is the amount of electrical stimulation necessary to express a therapeutically effective amount of an angiogenic protein in an amount to provide a therapeutic benefit or provides at least one measurable improvement in circulation.
• pace as used herein is the act of issuing an electrical subthreshold stimulus delivered to the cellular tissue delivered a subthreshold pulse generator.
• Puling generally refers to the act of repeatedly issuing an electrical stimulation to the tissue, as in the present case, delivering a series of subthreshold stimulations to the tissue.
• “Pharmacologically effective stimulus” is the amount of stimulus needed to provide a desired level of an angiogenic protein in the patient. The precise amount of stimulation or pacing needed will depend upon numerous factors, e.g., such as the specific angiogenic factor involved, the delivery stimulus employed, characteristics of stimulus provided, its intended use, and patient considerations. These determinations can readily be determined by one skilled in the art in view of the information provided herein.
• administer an electrical stimulus means to deliver electrical stimulation to a tissue.
• the electrical stimulus is delivered to the tissue by a subthreshold pulse generator.
• Threshold versus “subthreshold” stimulation refers to a relative level of applied stimulation.
• Threshold stimulation as used herein, refers to a level of stimulation to evoke a gross tissue electrical or mechanical response in the excited tissue, e.g. the minimum electrical stimulus needed to consistently elicit a cardiac depolarization for a heart contraction or to elicit a skeletal muscle movement.
• threshold stimulation is greater than 1.0 volt.
• Subthreshold stimulation refers to the application of electrical stimulation to tissue at levels low enough not to elicit a gross electrical or mechanical response from the tissue, such as to not cause cardiac depolarization or muscle contraction.
• a subthreshold stimulus can be achieved by keeping either the voltage amplitude and/or the duration of the electrical pulses below the threshold response levels for gross motor or nerve responses. Generally, subthreshold stimulus is less than or equal to 1.0 volt.
• Subthreshold stimulation allows one to deliver electrical stimulation to the tissue to increase the levels of angiogenic protein available without having the unwanted side effects due to the stimulation of nerve or muscle cells, such as unwanted contraction and or uncomfortable tactile sensations, and the like.
• amplitude may be expressed in volts (V) or millivolts (mV); current may be expressed in amperes (amps) or milliamperes (mamps); and pulse width, frequency, or timing in milliseconds (msec); and energy in joules (J) or millijoules (mJ).
• the present invention relates to a subthreshold pulse generator for producing an electrical field near to or within a targeted body tissue and methods of treating damaged or ischemic tissue by stimulating angiogenesis.
• the electrical field is delivered directly to the target area located between a plurality of electrodes.
• Figure 1 (Fig. 1) illustrates a subthreshold pulse generator (1) creating a subthreshold electrical field (5) to the lower ventricle of the heart (4) through a pair of leads (2) and electrodes (3).
• Subthreshold stimulation has been demonstrated to promote production of angiogenic growth factors.
• the promotion of angiogenic response by the present device thereby serves as a significant adjunct over current surgical methods of intervention to re-establish circulation to ischemic tissue areas.
• the present invention provides a novel subthreshold electrical pulse generator (also referred to herein as subthreshold pulse generator, pulse generator, or generator).
• the pulse generator has the essential feature of being capable of providing an electrical stimulus or series of electrical subthreshold stimulations or pulses (pacing).
• the subthreshold electrical stimulus or pulses are used to induce angiogenesis in targeted cells or tissues.
• the electrical stimulator provides a subthreshold stimulation to activate transcription of at least one angiogenic factor.
• the objective of the subthreshold stimulation is not to excite the tissue for mechanical contraction but to selectively activate angiogenesis. It is envisioned that different stimulation therapies may be given in conjunction with a course of subthreshold stimulation therapy. At times, particularly when considered with benefits of threshold electrical stimulation of traditional pacemakers, it may be advantageous to combine the features of a traditional pacemaker with components for subthreshold stimulation.
• the controlled output voltage from the subthreshold electrical pulse generator can be adjusted for a wide range of tissue impedances, such as from 35 ⁇ to infinity.
• tissue impedances such as from 35 ⁇ to infinity.
• the present generator allows the use of a constant voltage, regardless of the distance between electrodes, by allowing a variable field density.
• the present generator allows for control over the amplitude of the voltage and for charge balancing of the delivered and recovered charged pulse.
• the subthreshold pulse generator allows the use of a constant voltage by allowing a variable field density of about 30% of the targeted voltage, more preferably of about 20% of the targeted voltage, and even more preferably of about 10%, and most preferably of about 5% of the targeted voltage.
• the subthreshold voltage is at a constant level, allowing the field intensity to vary across the tissue. Effective results were obtained in the in vitro experiments having a variable field intensity in vitro (see Experiment 1, Table 1) as well as with stimulating VEGF production in vivo (see Experiment 2, Figure 7).
• the subthreshold pulse generator is capable of delivering an electric field to the targeted body tissue of 0 to 1.5 V output in steps of 0.1 V, wherein the electric field is generally less than or equal to about 1 V/cm, and more preferably less than about .5 VI cm, and even more preferably about 0.1 V/cm.
• the subthreshold field can be produced by a number of pulses with a frequency about 10 Hz to about 100 Hz, and preferably with a frequency of about 25 Hz to about 85 Hz, and more preferably with a frequency of about 40 Hz to about 70 Hz, and even more preferably with a frequency of about 50 Hz to about 60 Hz, and most preferably with a frequency of about 50 Hz.
• the stimulation period is less than the pulse cycle (1 /frequency).
• the stimulation period can be chosen to be between about 100 msec and .01 msec, between about 50 msec and .05 msec, and between 3 msec and .1 msec, wherein the actual value can be less than about 20 msec, preferably less than about 10 msec, more preferably less than about 3 msec or any value between 0.1ms and 3.0 ms.
• FIG. 2 is a simplified illustration of the schematic of the operating circuitry for a subthreshold pulse generator.
• Fig. 2 is useful for setting basic design requirements of a subthreshold pulse generator: S l5 S 2 , and S 3 are switches that are opened and closed during the operating cycle, R is a circuit resistor, Vs is the battery, and C H and C c are capacitors.
• S l5 S 2 , and S 3 are switches that are opened and closed during the operating cycle
• R is a circuit resistor
• Vs is the battery
• C H and C c are capacitors.
• FIG. 3 shows the equivalent circuit of the output stage during the stimulation phase.
• V c represents the initial condition on the C H .
• 0.090 0.110 ⁇ 1 - exp [-t / ( C Eq R Tissue )] ⁇
• t 0.3msec
• the minimum tissue impedance that one can drive will be 35 ⁇ , with output voltage staying in the 90-110 mV range.
• Use of the above settings in the pulse generator provides one example for (1) a pulse generator for subthreshold stimulation; (2) controlled output voltage for a wide range of tissue impedances (35 ⁇ to infinity); (3) a pacing output for subthreshold stimulation where the objective is not to excite the tissue for mechanical contraction but to increase the amounts of angiogenic factors available by providing a set of variables equal to or less than a 1.0 volt subthreshold stimulus.
• Figure 4 (Fig. 4) exemplifies a set of subthreshold stimulation parameters.
• Fig 4. illustrates the timing diagram of the electrical pulses and charging of capacitors, to provide the illustrated pulse train: pulse frequency of 50Hz (20 msec); stimulatory pulse of 0.3 msec to the tissue (Vtissue) and a 6.7 msec discharge (recharge with opposite polarity for charge balance). The remaining 13 msec of the pulse cycle electrodes are floating, and capacitors are recharged.
• FIG. 5 is a block diagram of a subthreshold stimulator employed in chronic animal studies.
• the basic stimulus repetition frequency of 50 Hz is generated by the clock/timer at upper left. This triggers timers that control the 1 msec discharge and 5 msec recharge phases of each output pulse (top center). Pulse amplitude is controlled by 4-bit DAC (left center).
• the pulse output circuit (center) utilizes the timing and amplitude information to generate the actual output pulse which is, in turn, delivered to electrodes and the tissue (right center).
• the pulse output circuit (bottom center) optionally incorporates a lead continuity monitor to check for lead or electrode malfunction.
• Output amplitude may be adjusted (dashed line) based on conditions of increased electrode resistance, or turned off if a lead breaks or shorts.
• An alternative stimulation setting of 2 Hz (upper left) is employed for evaluation of the stimulation safety margin (pacing threshold) in conjunction with the output amplitude control.
• FIG. 6 shows the stimulator circuit used during in vivo experiments.
• Explanations of general symbols used in the circuit diagram are as follows: U: Integrated Circuit R: Resistor C: Capacitor
• Vcc Positive terminal of the power supply (battery)
• Vee Negative terminal of the power supply (battery)
• JP Jumper terminal for off-board connections
• CIO is the coupling capacitor, C c , with the value of 6.8 micro-
• JP2 is the header where the stimulation electrodes are attached.
• U5 Switch S3, when closed discharges the coupling capacitor.
• R9 Resistor in series with the tissue being stimulated that is used to measure the stimulation current intensity.
• U7C Digital to analog converter which is used to set the stimulation amplitude, Vadj, shown as (14).
• U7D Inverter/driver for the adjustable stimulation amplitude determined by (11).
• Vadj adjustable stimulation amplitude determined by (11)
• the subthreshold stimulator circuit of Figure 6 A (Fig 6 A) and continued on Figure 6B (Fig. 6B) operates essentially to produce the stimulation waveform shown in Fig. 2.
• the generated stimulus is a periodic signal which main oscillator (1) is set to produce 50 Hz digital pulses, and provides the main clock.
• Timers (2) and (3) produce the stimulation and discharge pulses, respectively, from the clock, again as shown on Figure 2. These pulses are used to close the switches (13) and (7), which correspond to S 2 and S 3 on Figure 2.
• Terminals 1 and 4 of the on board connector (6) is where the leads going to the tissue are attached.
• energy stored on holding capacitor (6) passes through switch (13), the coupling capacitor (5), and series resistor (8) to reach terminal (6) before arriving at the tissue.
• Voltage drop on series resistor (8) can be monitored from terminal (6) to get an indication of the current being passed through the tissue.
• Switch (9) can be used to monitor the lead integrity using the lead integrity indicator (10).
• Stimulation amplitude adjuster (14) is set by the digital to analog converter (11) followed by the inverter/driver (12).
• the subthreshold pulse generator can be used externally, but preferably is designed and configured to be implantable.
• the present device can be implanted into the body and the electrical components sealed from the body tissues and fluids.
• the implantable device has a volume of about 50 cm 3 , preferably about 40 cm 3 , more preferably about 30 cm 3 , even more preferably about 20 cm 3 , and most preferably about 10 cm 3 .
• the electrical pulse generator can be implanted or can be external to the body. Ideally, the subthreshold pulse generator is implanted.
• the subthreshold pulse generator includes a power supply and a control mechanism interconnected with the power supply.
• the pulse generator can be used with electrodes in electrical communication with the power supply.
• he subthreshold stimulation provided is sufficient to stimulate angiogenesis in the targeted body tissue.
• the present generator is also capable of checking the lead continuity at a predesignated time, hi other preferred embodiments, electrodes and leads can be used with the subthreshold pulse generator.
• the electrodes are configured in a manner consisting of bipolar or multiple electrode configurations.
• the electrodes are made of conductive metals or organic polymers, or combinations of the two.
• the electrodes can be made of platinium, gold, zirconium, iridium, titanium, certain carbons, stainless steel, silver, copper, tin, nickel, iron, or lithium, or various mixtures, alloys, or amalgams thereof.
• Design of the electrodes can take on a number of different shapes and sizes, depending on the nature of the target tissue. In the case of heart muscle or other muscle tissues, the electrodes can consist of a straight pin, screw, patch, or the like, which can further comprise various barbs, hooks, or alternate structures for affixing the electrode.
• various types of electrical leads similar to those exemplified herein or commonly used with other implantable pulse generators can be used to connect to the power source.
• a number of suitable electrodes can function to provide the electrical stimulation.
• the electrode is a surface coil electrode, or heart wire.
• the surface electrode may be constructed of a platinum alloy or other biocompatible metals.
• the electrode can be a coil, a cylinder, a wire, or any other shape.
• Electrode placement can be done in one of two ways: In the preferred embodiment, electrodes are advanced to the vicinity of the tissue of the heart where the angiogenesis is desired, using the venous system, and left in place. Alternatively, it is possible to place the electrodes in place using minimally invasive surgical procedures, which would allow access to locations that are beyond the reach of the catheters in the vasculature. In either case, bipolar or unipolar stimulation can be applied to generate the electrical fields in the tissue to trigger the electrically responsive promoter. Bipolar stimulation is the preferred method.
• the placement of the electrodes would be determined primarily by the method used to implant the electrodes. If the electrodes are placed via a transvenous route then the electrodes should be placed as close as possible to the implanted cells, ischemic tissue, or target area for angiogenesis, understanding that patient anatomy may not allow close proximity of the electrodes. If a non-transvenous implant technique is used, then the stimulating electrodes can usually be placed very close to the ischemic area.
• the subthreshold stimulation provided is sufficient to stimulate angiogenesis in the targeted body tissue of a patient.
• the pulse generator is used to modulate transcription of angiogenic growth factors by the delivery of subthreshold electrical fields.
• the invention also provides a subthreshold pulse generator for a patient in need of subthreshold stimulation therapy.
• the invention includes a method for reducing or repairing tissue injury by providing a means for regulating angiogenic growth factor production.
• the pulse generator is effective in delivering subthreshold pulses that mediate the repair of injured muscle tissue, such as where ischemic injury has occurred.
• the method may be applied to damaged cardiac or peripheral muscle tissue by providing a therapeutic stimulus to the surrounding cardiac or muscle tissue or cells.
• vascular muscle tissue is stimulated using the subthreshold pulse generator.
• Subthreshold stimulation of vascular tissue includes stimulation of arteries and veins in a patient.
• the present system can be used to treat peripheral arterial occlusive disease (PAOD) or coronary arterial disease (CAD) or stroke, by delivery of a therapeutically effective amount of subthreshold stimulation. It is envisioned that treatment of peripheral arterial occlusive disease (PAOD) or coronary arterial disease (CAD) is achieved by stimulation of angiogenic proteins, such as VEGF and FGF, to enhance blood vessel formation (angiogenesis).
• PAOD peripheral arterial occlusive disease
• CAD coronary arterial disease
• the present invention also provides a novel method of stimulating cells for controlled expression of angiogenic factors.
• the stimulated cells are muscle cells, hi another preferred embodiment the cells are muscle cells, and more preferably, heart, smooth, or skeletal muscle cells.
• subthreshold pulses are proved to enhance the cellular production of endogenous angiogenic growth factors of the transplanted cells.
• the present device can be used in vitro to pre-stimulate cells which may then be transplanted to the heart. In this process, cells in culture are stimulated in a subthreshold field and used for transplantation. In this process cells may be taken from the patient (autologous cell transplantation) or used from a different patient of the same species (allogenic cell transplantation) or from a different species (xenogenic cell transplantation).
• Transplanted cells or grafts may be derived from auto-, allo- or xeno-graphic sources.
• Transplanted or grafted cells for heart tissue used with pre- or post subthreshold stimulation can be chosen from the group consisting of adult cardiomyocytes, pediatric cardiomyocytes, fetal cardiomyocytes, adult fibroblasts, fetal fibroblasts, adult smooth muscle cells, fetal smooth muscle cells, endothelial cells, and skeletal myoblasts (see US patent No. 6,099,832 and procedures described herein for isolation of various cell types). A number of additional procedures are known and described in the art for isolating various primary cell types.
• Human VEGF in samples was quantified using the Human VEGF Immunoassay by Quantikine®. The protocol followed was essentially as described in the Quantikine® Catalog (Number DVEOO).
• the Human VEGF Immunoassay employs a sandwich enzyme immunoassay technique. A monoclonal antibody specific for VEGF is pre-coated on micro- titer plates. Standards and samples are pipetted into wells and any VEGF present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme- linked polyclonal antibody specific for VEGF is added to the wells.
• Example 1 Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of VEGF bound in the initial step. The color development is stopped and the intensity of the color is measured.
• Example 1 Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of VEGF bound in the initial step. The color development is stopped and the intensity of the color is measured.
• Example 1 Example 1 :
• Sterile 6 well culture plates were seeded with cells on six well culture inserts using SmGM growth media; C 2 C 12 cells (mouse myoblasts) were seeded at 7.5 x 10 3 /cm 2 ; Human Coronary Smooth Muscle Cells (HCASMC) were seeded at 2.5 x lOVcm 2 .
• HCASMC Human Coronary Smooth Muscle Cells
• the wells were washed twice with electrical stimulation medium (DMEM with 1% bovine serum albumin). 2.0 ml of serum free medium was added but without any fetal bovine serum in the medium.
• Wells contained approximately 4 ml of total growth medium, approximately 2.0 ml inside well insert and 2.0 ml outside well insert.
• the cells were electrically stimulated using a circular graphite electrode for 8 hours per stimulation condition. Cells were stimulated at one volt in the stimulation chamber for 1 msec stimulation pulse width with a 4 msec discharge pulse width. The escape period was adjusted to achieve the desired frequency. Samples were harvested after 22 hours post-stimulation. Cell culture supernatants were removed from the well. Any debris or floating cells were removed by centrifuging the supernatants at 300 RPM for 5 minutes prior to freezing the samples at -85° C. A cell count was done on all wells of the culture plate.
• Example 2 In Vivo Subthreshold Stimulation In Canine Model of Regional Ischemic Cardiomyopathy.
• Dogs were initially anesthetized with intramuscular morphine sulfate (4 mg/kg)/ A bolus injection of pentothal (20 mg/kg) was given followed by continuous inhaling of isoflurane (0.5%-2% in oxygen) after endotracheal intubation. A left lateral thoracotomy was performed, and the pericardium opened. A micromanometer pressure transducer (MPC-500, Millar Instruments, Houston, Texas) was inserted into the left ventricle through an apical incision. Pairs of 5 MHz ultrasonic crystals also was implanted in the area supplied by the distal left anterior descending (LAD) and left circumflex (LCX) arteries just distal to the first diagonal branch of measurement of coronary flow.
• LAD distal left anterior descending
• LCX left circumflex
• Two heart wire electrodes were inserted in the LAD perfusion area.
• a catheter was inserted into the left atrium for injection of colored 15 um microspheres to measure regional myocardial blood flow.
• Ameriod constrictors were placed on the LAD artery proximal to the flow probe. All wires and tubing were tunneled subcutaneously and brought out through the skin of the dorsal neck. The thoracotomy incision was closed in layers, and a regimen of broad spectrum antibiotics and analgesia was initiated throughout the reperfusion period.
• the basic chronology of the experimental protocol after surgery was divided into three periods.
• the first period (recovery period) occurred in the first week after surgery. After surgery to instrument the dogs, the dogs were allowed to recover.
• the second period occurred during weeks 2 through 5 after surgery, and included weekly monitoring of regional stroke work, CBF (LAD and LCX), and left ventricular pressure (LVP) in addition to microsphere injections for evaluating resting CBF.
• the potential loss of microspheres from chronic postischemic tissue is corrected by using a rate factor of baseline flow in nonischemic tissue applied to ischemic tissue blood flow data (e,g, ischemic/nonischemic flow).
• ischemic tissue blood flow data e,g, ischemic/nonischemic flow.
• subthreshold pulsing was delivered to the heart using a subthreshold stimulator operated at 50Hz, 0.1V, with 0.3 ms pulses.
• the subthreshold stimulator had an operational range of 0 to 1.5 V output in steps of 0.1 V for all 16 allowed settings, pulse widths of 0.1, 0.3, 1.0, and 3.0 m by a series of 4 slide switches.
• the stimulator also had the ability to determine pacing threshold and thus the margin or extent to which the 0.1V, 50Hz pulses are subthreshold. This is done by setting the device to an assessment mode of 1-3 Hz while stepping up the amplitude to look for VOO pacing capture. Stimulation was delivered via a set of myocardial pacing wires or "Heart- Wires" that were connected to the stimulator through a set of unipolar IS-1 leads to abiopolar IS-1 connector.
• the pulse generator contains a combination battery level OK and pacing wire continuity OK indicator with a visible LED light.
• the LAD heart area was stimulated for 5 days during week six before terminating the experiment.
• Sample heart tissue was collected from both stimulated and unstimulated dogs.
• Heart tissue samples were taken and prepared for Western blot analysis using a VEGF antibody (Fig. 7).
• Total protein was extracted from postexperimental cardiac tissue. 100 ug of protein was loaded onto each lane of a SDS-Page gel, and run for 15 minutes at 200 Volts. The lanes were exposed to a polyclonal antibody for human VEGF (Santa Cruz). This antibody was blocked by a molecule that blocks the antibody's binding epitope (data not shown), and hence is specific for the VEGF site.
• Lane 8 - 11 control group.
• Lane 8 nonischemic transmural
• Lane 9 ischemic subepicardium
• Lane 10 ischemic subendocardium
• Lanes 12 - 15 are treatment (field stimulation) group: Lane 12 (transmural nonischemic); Lane 13 (ischemic subepicardium); Lane 14 (ischemic subendocardium); Lane 15 (right ventricle).
• Bands for VEGF appear at approximately 26 kD molecular weight. Two bands appear, typical of the two fragments seen for VEGF. Notice that bands consistent with VEGF appear only in Lanes 12 - 15, i.e. in the treated group. Gels were processed on the same day to avoid variability.

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