Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.     

Intramyocardial Injections to De-Stiffen the Heart: A Subject-Specific in Silico Approach

We hypothesized that minimally invasive injections of a softening agent at strategic locations in stiff myocardium could de-stiffen the left ventricle (LV) globally. Physics-based finite element models of the LV were created from LV echocardiography images and pressures recorded during experiments in four swine. Results confirmed animal models of LV softening by systemic agents. Regional de-stiffening of myocardium led to global de-stiffening of LV. The mathematical set up was used to design LV global de-stiffening by regional softening of myocardium. At an end diastolic pressure of 23 mmHg, when 8 ml of the free wall was covered by intramyocardial injections, end diastolic volume (EDV) increased by 15.0%, whereas an increase up to 11 ml due to intramyocardial injections in the septum and free wall led to a 26.0% increase in EDV. Although the endocardial intramyocardial injections occupied a lower LV wall volume, they led to an EDV (44 ml) that was equal compared to intramyocardial injections in the mid-wall (44 ml) and larger compared to intramyocardial injections in the epicardium (41 ml). Using an in silico set up, sites of regional myocardium de-stiffening could be planned in order to globally soften overly stiff LV in heart failure with preserved ejection fraction. This novel treatment is built on subject-specific data. Hypothesis-testing of these simulation findings in animal models is warranted.     

Immuno‐modification of enhancing stem cells targeting for myocardial repair

Despite the controversy in mechanism, rodent and clinical studies have demonstrated beneficial effects of stem/progenitor cell therapy after myocardial infarction (MI). In a rat ischaemic reperfusion MI model, we investigated the effects of immunomodification of CD 34⁺ cells on heart function and myocardial conduction. Bispecific antibody (BiAb), consisting of an anti‐myosin light chain antibody and anti‐CD45 antibody, injected intravenously was used to direct human CD34⁺ cells to injured myocardium. Results were compared to echocardiography guided intramyocardial (IM) injection of CD34⁺ cells and PBS injected intravenously. Treatment was administered 2 days post MI. Echocardiography was performed at 5 weeks and 3 months which demonstrated LV dilatation prevention and fractional shortening improvement in both the BiAb and IM injection approaches, with BiAb achieving better results. Histological analyses demonstrated a decrease in infarct size and increase in arteriogenesis in both BiAb and IM injection. Electrophysiological properties were studied 5 weeks after treatments by optical mapping. Conduction velocity (CV), action potential duration (APD) and rise time were significantly altered in the MI area. The BiAb treated group demonstrated a more normalized activation pattern of conduction and normalization of CV at shorter pacing cycle lengths. The ventricular tachycardia inducibility was lowest in the BiAb treatment group. Intravenous administration of BiAb offers an effective means of stem cell delivery for myocardial repair post‐acute MI. Such non‐invasive approach was shown to offer a distinct advantage to more invasive direct IM delivery.     

Study of the impact of intramyocardial implantation of stem cells on myocardial perfusion and contractility in patients with coronary heart disease concurrent with postinfarct cardiosclerosis and chronic heart failure

OBJECTIVE: to study of intramyocardial implantation of cultured bone marrow stem cells on myocardial perfusion and contractility in the surgical treatment of patients with coronary heart disease (CHD) and chronic heart failure (CHF), by synchronized single-photon emission computed tomography (SSPECT) of the myocardium. SUBJECTS AND METHODS: The study included 11 patients. Intramyocardial injection of cell injections into the myocardial periscarring areas was made at coronary bypass surgery. All the patients underwent 99mTc myocardial SSPECT MIBI before and 3, 6, 12 months after surgery. RESULTS AND CONCLUSIONS: Implantation of bone marrow stem cells into the left ventricular myocardium favorably affects left ventricular remodeling and contributes to the improvement of myocardial perfusion and contractility, as evidenced by 99mTc.     

The effects of afterload and stimulation delay on the slow force response in the heterogeneous myocardium

The results of numerical simulations and physiological experiments that assessed the dependence of the intramyocardial slow force response under an afterload in the heterogeneous myocardium are described. Muscle duplexes of our design were used as the simplest experimental and theoretical models of the heterogeneous myocardium. It is shown that the degree of the slow force response increases with an afterload and the time of the stimulation delay between the elements of the duplex. This effect is explainable by an increase in the time of the mechanical interaction between the duplex elements in the isometric phase of contraction. This leads to changes in individual contractility, the configuration of the action potential, and the kinetic characteristics of intracellular calcium in cardiomyocytes of interacting muscles.     

Enhanced infarct myocardium repair mediated by thermosensitive copolymer hydrogel-based stem cell transplantation

Mesenchymal stem cell (MSC) transplantation by intramyocardial injection has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, low retention and survival of grafted MSCs hinder its further application. In this study, copolymer with N-isopropylacrylamide/acrylic acid/2-hydroxylethyl methacrylate-poly(ɛ-caprolactone) ratio of 88:9.6:2.4 was bioconjugated with type I collagen to construct a novel injectable thermosensitive hydrogel. The injectable and biocompatible hydrogel-mediated MSC transplantation could enhance the grafted cell survival in the myocardium, which contributed to the increased neovascularization, decreased interstitial fibrosis, and ultimately improved heart function to a significantly greater degree than regular MSC transplantation. We suggest that this novel hydrogel has the potential for future stem cell transplantation.     

Improvement in the severity of mitral regurgitation after direct intramyocardial injection of angiogenic cell precursor

We reported a case of dilated cardiomyopathy and moderate-severe mitral regurgitation (MR) who we treated by surgical direct intramyocardial angiogenic cell precursors injection. The patient was a New York Heart Association functional class III-IV, 56 year old man, who presented with end-stage congestive heart failure, moderate/severe mitral regurgitation, and myocardial fibrosis with the left ventricular ejection fraction of 13%. After he underwent direct surgical intramyocardial cell implantation, the myocardial fibrosis was resolved at 3 months follow-up. The severity of MR reduced to moderate and mild at 3 and 9 months, respectively. The left ventricular function gradually improved up to 53% at 19 months. To our knowledge, this is one of the only reports of successful direct surgical intramyocardial peripheral blood stem cell implantation to treat MR in dilated cardiomyopathy patient.     

Active and passive stresses in the myocardium

A mathematical approach that can be used to calculate the passive stress in the ventricular wall is presented. The active fiber stress (force/unit area) generated by the muscular fibers in the ventricular wall is expressed by means of body force (force/unit volume of the myocardium). It is shown that the total intramyocardial passive stress induced in the passive medium of the myocardium can be expressed as the sum of a passive stress induced by the left ventricular pressure and a passive stress induced by the active fiber stress. Applications to experimental data published in the literature are given. New results are presented that show the relation among those two components of the intramyocardial passive stress. New relations between the intramyocardial passive stress, the slope (elastance) of the pressure-volume relation, and the residual volume are also derived. The results obtained give a better understanding of some aspects of the mechanics of cardiac contraction and can provide a more detailed interpretation of clinical conditions.     

Selective freezing of target biological tissues after injection of solutions with specific thermal properties

Cryosurgery is a minimally invasive surgical technique that employs the destructive effect of freezing to eradicate undesirable tissues. This paper proposes a flexible method to control the size and shape of the iceball by injecting solutions with specific thermal properties into the target tissues, to enhance freezing damage to the diseased tissues while preserving the normal tissues from injury. The cryosurgical procedure was performed using a minimally invasive cryoprobe cooled by liquid nitrogen (LN2) to obtain deep regional freezing. Several needle thermocouples were applied simultaneously to record the transient temperature to detect the freezing effect on the tissues. Simulation experiments on biological tissue (fresh pork) were performed in vitro and four different liquids were injected into the test materials; these were distilled water, an aqueous suspension of aluminum nanoparticles in water, ethanol, and a 10% solution of the cryoprotective agent dimethyl sulfoxide (Me2SO). The experimental results demonstrate that the localized injection of an appropriate solution could enhance the tumor-killing effect without altering the freezing conditions. The study also suggests the potential value of combining cryosurgery with other therapeutic methods, such as electrical, chemical, and thermal treatments, to develop new clinical modalities in the near future.     

Cardiomyogenic differentiation-independent improvement of cardiac function by human cardiomyocyte progenitor cell injection in ischaemic mouse hearts

We previously showed that human cardiomyocyte progenitor cells (hCMPCs) injected after myocardial infarction (MI) had differentiated into cardiomyocytes in vivo 3 months after MI. Here, we investigated the short-term (2 weeks) effects of hCMPCs on the infarcted mouse myocardium. MI was induced in immunocompromised (NOD/scid) mice, immediately followed by intramyocardial injection of hCMPCs labelled with enhanced green fluorescent protein (hCMPC group) or vehicle only (control group). Sham-operated mice served as reference. Cardiac performance was measured 2 and 14 days after MI by magnetic resonance imaging at 9.4 T. Left ventricular (LV) pressure-volume measurements were performed at day 15 followed by extensive immunohistological analysis. Animals injected with hCMPCs demonstrated a higher LV ejection fraction, lower LV end-systolic volume and smaller relaxation time constant than control animals 14 days after MI. hCMPCs engrafted in the infarcted myocardium, did not differentiate into cardiomyocytes, but increased vascular density and proliferation rate in the infarcted and border zone area of the hCMPC group. Injected hCMPCs engraft into murine infarcted myocardium where they improve LV systolic function and attenuate the ventricular remodelling process 2 weeks after MI. Since no cardiac differentiation of hCMPCs was evident after 2 weeks, the observed beneficial effects were most likely mediated by paracrine factors, targeting amongst others vascular homeostasis. These results demonstrate that hCMPCs can be applied to repair infarcted myocardium without the need to undergo differentiation into cardiomyocytes.     

Refinement of intrathymic injection in mice

Direct intrathymic injection is a common procedure used in several types of experimental protocols in the mouse. Currently available approaches involve major surgical procedures that expose the thoracic cavity, resulting in an increased risk of poor recovery and postsurgical complications. The authors sought to refine this surgery to reduce animal pain and distress without compromising overall efficiency of the technique. Using a minimally invasive method that does not expose the thoracic cavity, the authors gave accurately placed intrathymic injections, as confirmed by analyses with a reporter dye. They describe this new approach for intrathymic injection in mice that reduces complications associated with lengthy periods of anesthesia and thoracic cavity exposure.     

Closed-chest cell injections into mouse myocardium guided by high-resolution echocardiography

The mouse is an important model for the development of therapeutic stem cell/bone marrow cell implantation to treat ischemic myocardium. However, its small heart size hampers accurate implantation into the left ventricular (LV) wall. Precise injections have required surgical visualization of the heart, which is subject to complications and is impractical for delayed or repeated injections. Furthermore, the thickness of the myocardium is comparable to the length of a needle bevel, so surgical exposure does not prevent inadvertent injection into the LV cavity. We describe the use of high-resolution echocardiography to guide nonsurgical injections accurately into the mouse myocardial wall. We optimized this system by using a mixture of ultrasound contrast and fluorescent microspheres injected into the myocardium, which enabled us to interpret the ultrasound image of the needle during injection. Quantitative dye injection studies demonstrated that guided closed-chest injections and open-chest injections deliver comparable amounts of injectate to the myocardium. We successfully used this system in a mouse myocardial infarction model to target the injection of labeled cells to a region adjacent to the infarct. Intentional injection of tracer into the LV cavity resulted in a small accumulation in the myocardium, suggesting that non-guided cell injections into mouse hearts may appear to be successful even if the majority of the injectate is lost in the chamber. The use of this system will allow more precise cellular implantation into the mouse myocardium by accurately guiding injections to desired locations, confirming successful implantation of cells, in a clinically relevant time frame.     

Oxygen-dependent quenching of phosphorescence used to characterize improved myocardial oxygenation resulting from vasculogenic cytokine therapy

This study evaluates a therapy for infarct modulation and acute myocardial rescue and utilizes a novel technique to measure local myocardial oxygenation in vivo. Bone marrow-derived endothelial progenitor cells (EPCs) were targeted to the heart with peri-infarct intramyocardial injection of the potent EPC chemokine stromal cell-derived factor 1α (SDF). Myocardial oxygen pressure was assessed using a noninvasive, real-time optical technique for measuring oxygen pressures within microvasculature based on the oxygen-dependent quenching of the phosphorescence of Oxyphor G3. Myocardial infarction was induced in male Wistar rats (n = 15) through left anterior descending coronary artery ligation. At the time of infarction, animals were randomized into two groups: saline control (n = 8) and treatment with SDF (n = 7). After 48 h, the animals underwent repeat thoracotomy and 20 μl of the phosphor Oxyphor G3 was injected into three areas (peri-infarct myocardium, myocardial scar, and remote left hindlimb muscle). Measurements of the oxygen distribution within the tissue were then made in vivo by applying the end of a light guide to the beating heart. Compared with controls, animals in the SDF group exhibited a significantly decreased percentage of hypoxic (defined as oxygen pressure ≤ 15.0 Torr) peri-infarct myocardium (9.7 ± 6.7% vs. 21.8 ± 11.9%, P = 0.017). The peak oxygen pressures in the peri-infarct region of the animals in the SDF group were significantly higher than the saline controls (39.5 ± 36.7 vs. 9.2 ± 8.6 Torr, P = 0.02). This strategy for targeting EPCs to vulnerable peri-infarct myocardium via the potent chemokine SDF-1α significantly decreased the degree of hypoxia in peri-infarct myocardium as measured in vivo by phosphorescence quenching. This effect could potentially mitigate the vicious cycle of myocyte death, myocardial fibrosis, progressive ventricular dilatation, and eventual heart failure seen after acute myocardial infarction.     

Expression of matrix metalloproteinase-1 (MMP-1) in Wistar rat's intervertebral disc after experimentally induced scoliotic deformity

Minimally invasive spine surgery is becoming more common in the treatment of adult lumbar degenerative disorders. Minimally invasive techniques have been utilized for multilevel pathology, including adult lumbar degenerative scoliosis. The next logical step is to apply minimally invasive surgical techniques to the treatment of adolescent idiopathic scoliosis (AIS). However, there are significant technical challenges of performing minimally invasive surgery on this patient population. For more than two years, we have been utilizing minimally invasive spine surgery techniques in patients with adolescent idiopathic scoliosis. We have developed the present technique to allow for utilization of all standard reduction maneuvers through three small midline skin incisions. Our technique allows easy passage of contoured rods, placement of pedicle screws without image guidance, and allows adequate facet osteotomy to enable fusion. There are multiple potential advantages of this technique, including: less blood loss, shorter hospital stay, earlier mobilization, and relatively less pain and need for pain medication. The operative time needed to complete this surgery is longer. We feel that a minimally invasive approach, although technically challenging, is a feasible option in patients with adolescent idiopathic scoliosis. Although there are multiple perceived benefits, long term data is needed before it can be recommended for routine use.     

Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium

We hypothesised that angiopoietin-1 (Ang-1), in conjunction with vascular endothelial growth factor (VEGF) gene therapy, can enhance arteriogenesis and angiogenesis during myocardial ischemia. Mice were given a single intramyocardial injection of saline, phVEGF-A(165) and phAng-1 or a combination thereof into the non-ischemic normal heart or into the ischemic border zone of the infarcted heart. In the normal and the ischemic myocardium, gene transfer of phVEGF-A(165) alone increased the myocardial capillary density by 16% and 36%, respectively, and phAng-1 had a similar effect. In the normal heart, the ratio of arteriolar to capillary densities increased with phVEGF-A(165) and more so in the ischemic myocardium where phAng-1 also had an effect. Furthermore, the combination of plasmids induced an up to 7.5-fold increase. Transient overexpression of VEGF-A(165) boosts endogenous arteriogenesis in addition to capillary angiogenesis. Ang-1 further boosts this effect at the arteriolar level.     

Catheter-based transendocardial gene delivery for therapeutic myocardial angiogenesis

Currently it is unknown which is the most effective and safest delivery strategy for therapeutic myocardial angiogenesis. The enhancing effect of direct intramyocardial injection of angiogenic factors using either open-chest or catheter-based approaches on collateral function has been reported to occur in experimental models and among patients. This report reviews the rationale and current experience of using a catheter-based approach as a promising platform for proangiogenic intramyocardial gene delivery strategy.     

Direct cardiac injection of G-CSF mobilized bone-marrow stem-cells improves ventricular function in old myocardial infarction

Autologous transplant of bone marrow stem cells (BMSC), although extremely useful after acute myocardial events, has not been evaluated in patients with old (>one-year-old) myocardial infarction. Our aim was to determine if CD34(+)-enriched peripheral-blood cells, obtained by apheresis, injected directly into the severely damaged myocardium of five patients with old myocardial infarction could restore depressed myocardial function. We found that 28 weeks after revascularization and peri-infarction injection of the enriched CD34(+) peripheral mononuclear cells, ventricular hemodynamic parameters that included left ventricular ejection fraction, left ventricular diastolic volume, ventricular systolic volume and left ventricular diastolic diameter approximated normal values and there was no restenosis; two patients have been followed for >52 weeks and their parameters are within normal values. In conclusion, intramyocardial injection of easily obtained CD34(+) enriched peripheral blood cells represent an encouraging procedure for patients with severely scarred and dysfunctional myocardium.     

Flow and pressure responses of coronary arteries and veins to vasodilating agents

The response to a bolus injection of nitroglycerine, adenosine, nifedipine, and dipyridamole of the canine systemic as well as coronary artery and venous circulations was observed and contrasted. Particular attention was paid to the time of change of pressures and flows and to changes in oxygen extraction by the myocardium induced by the pharmacological agents. The dosages of vasodilators used were selected so that no significant change in aortic blood flow occurred. Nitroglycerine and adenosine caused a rapid and similar vasodilation in the coronary circulation. Oxygen extraction was not altered by nitroglycerine, but was decreased by adenosine. The onset time of the vasodilation produced by either nifedipine or dipyridamole was similar, but the time to peak action was much slower for dipyridamole. As well, the effect of dipyridamole on intramyocardial and left intraventricular pressures was more delayed than that following the injection of the other agents. Oxygen extraction was reduced by nifedipine and dipyridamole. These results indicate that pharmacological vasodilating agents can affect coronary arteries, coronary veins, and myocardial oxygen extraction differently.     

超声引导下麦默通微创旋切术在乳腺BI-RADS 3级肿块中的应用进展

麦默通微创旋切术(Mammotome)是目前临床上治疗乳腺BI-RADS 3级肿块常用的手术方式,其优势能够有效彻底清除病灶,而且术后创面恢复快、术口小.但微创手术也有出血、切破皮肤、感染以及病灶残留等相应并发症,可通过术中加入止血药物或者改良手术方式来减少并发症的发生几率.而针对麦默通微创旋切术后残留的热点问题,超声...     

Human endometrial stem cells confer enhanced myocardial salvage and regeneration by paracrine mechanisms

Human endometrial stem cells (EnSCs) have the potential to be ‘off the shelf’ clinical reagents for the treatment of heart failure. Here, using an immunocompetent rat model of myocardial infarction (MI), we provide evidence that the functional benefits of EnSC transplantation are principally and possibly exclusively through a paracrine effect. Human EnSCs were delivered by intramyocardial injection into rats 30 min. after coronary ligation. EnSC therapy significantly preserved viable myocardium in the infarct zone and improved cardiac function at 28 days. Despite increased viable myocardium and vascular density, there was scant evidence of differentiation of EnSCs into any cardiovascular cell type. Cultured human EnSCs expressed a distinctive profile of cytokines that enhanced the survival, proliferation and function of endothelial cells in vitro. When injected into the peri‐infarct zone, human EnSCs activated AKT, ERK1/2 and STAT3 and inhibited the p38 signalling pathway. EnSC therapy decreased apoptosis and promoted cell proliferation and c‐kit+ cell recruitment in vivo. Myocardial protection and enhanced post‐infarction regeneration by EnSCs is mediated primarily by paracrine effects conferred by secreted cytokines that activate survival pathways and recruit endogenous progenitor stem cells. Menstrual blood provides a potentially limitless source of biologically competent ‘off the shelf’ EnSCs for allogeneic myocardial regenerative medicine.