Clinical histopathology and ultrastructural analysis of myocardium following microwave energy ablation.

OBJECTIVE: Due to weaknesses of conventional modes for treating atrial fibrillation (AF), surgical energy ablation methods and tools to cure AF have been under rapid development. One of these methods, microwave energy, is beginning to be applied clinically. The purpose of this study was to examine histology and ultrastructure of lesions produced by microwave energy in the myocardium. METHODS: Fifteen consecutive patients underwent surgical microwave energy ablation (Microwave Ablation System with FLEX 4 probe, AFx Inc., Fremont, CA) concomitant to a valve procedure. Epicardial ablation was carried out on the beating normothermic heart prior to performing the valve procedure. Two tissue specimens (1cm(2)) were obtained from each patient; one from the lesion site (right appendage) and the other from an adjacent, non-ablated site, which was used as control. Tissue samples were fixed and stained as appropriate for histological and ultrastructural analysis. RESULTS: All ablated samples revealed observable microscopic alteration, including loss of nuclei, foci of coagulative necrosis or induced irregular bands of contraction. Ultrastructurally, ablated cells demonstrated architectural disarray, loss of contractile filaments, mitochondrial swelling and focal interruption of plasma membrane. CONCLUSIONS: Histologic appearance of lesions created by epicardial microwave energy ablation was consistent over tissue samples, although acute findings demonstrated differences from cryoablation. In most of the cases, lesions were transmural, as was demonstrated by loss of cellular viability throughout the depth of tissue specimens.     

TRB3 gene silencing alleviates diabetic cardiomyopathy in a type 2 diabetic rat model

OBJECTIVE

Tribbles 3 (TRB3) is associated with insulin resistance, an important trigger in the development of diabetic cardiomyopathy (DCM). We sought to determine whether TRB3 plays a major role in modulating DCM and the mechanisms involved.

RESEARCH DESIGN AND METHODS

The type 2 diabetic rat model was induced by high-fat diet and low-dose streptozotocin. We evaluated the characteristics of type 2 DCM by serial echocardiography and metabolite tests, Western blot analysis for TRB3 expression, and histopathologic analyses of cardiomyocyte density, lipids accumulation, cardiac inflammation, and fibrosis area. We then used gene silencing to investigate the role of TRB3 in the pathophysiologic features of DCM.

RESULTS

Rats with DCM showed severe insulin resistance, left ventricular dysfunction, aberrant lipids deposition, cardiac inflammation, fibrosis, and TRB3 overexpression. We found that the silencing of TRB3 ameliorated metabolic disturbance and insulin resistance; myocardial hypertrophy, lipids accumulation, inflammation, fibrosis, and elevated collagen I-to-III content ratio in DCM rats were significantly decreased. These anatomic findings were accompanied by significant improvements in cardiac function. Furthermore, with TRB3 gene silencing, the inhibited phosphorylation of Akt was restored and the increased phosphorylation of extracellular signal–regulated kinase 1/2 and Jun NH₂-terminal kinase in DCM was significantly decreased.

Conclusions.

TRB3 gene silencing may exert a protective effect on DCM by improving selective insulin resistance, implicating its potential role for treatment of human DCM.Diabetic cardiomyopathy (DCM), which occurs in patients with diabetes, carries a substantial risk concerning the subsequent development of heart failure and increased mortality (1). Different pathophysiological stimuli are involved in its development and mediate tissue injury leading to left ventricle (LV) systolic and diastolic dysfunction. Insulin resistance is considered to play a causal role in the pathogenesis and development of DCM (2–4). Insulin resistance is associated with increased LV mass (5) and deterioration of LV diastolic function (6). However, the underlying relevance of insulin resistance leading to altered myocardial structure remains incompletely understood.Tribbles 3 (TRB3) is a pseudokinase with increased activity in response to fasting that binds to and inhibits the activation of the serine-threonine kinase Akt in the liver (7,8). Indeed, humans with a gain-of-function mutation in TRB3 show increased insulin resistance and diabetes-associated complications (9,10). These observations have led to the suggestion that TRB3 elevation contributes to insulin resistance. TRB3 also serves as a molecular switch and regulates the activation of the three classes of mitogen-activated protein kinases (MAPKs) (11). TRB3 binds to and regulates MAPK kinase, thus controlling the activation of MAPK by incoming signals (11). However, the TRB3/MAPK signal-transduction pathway has not been investigated in vivo on cardiac tissues directly.Akt and MAPK are the most important pathways involved in selective insulin resistance (12), and activated MAPK contributes to the development of cardiac fibrosis (13–15). Thus, we hypothesized that upregulated TRB3 induced by insulin resistance might participate in the pathophysiological process of DCM. First, we established the type 2 DCM model and determined the relationships among TRB3 expression, cardiac remodeling, and cardiac function in the model. To further elucidate the role of TRB3 in DCM, we used TRB3 gene silencing in vivo to explore the mechanisms of TRB3 in DCM as a potential target for treatment.     

Effects of chlorpromazine on ischemic rat kidney: a functional and ultrastructural study

The effects of chlorpromazine, an agent with inhibitory effects of calcium influx, phospholipase activation, and Na-K-ATPase, on preserving renal function and proximal tubular ultrastructure were evaluated in renal ischemia. After right nephrectomy chlorpromazine (0.025 mg) or 1 ml of 0.9 per cent saline was selectively administered to the rat kidney immediately prior to a sixty-minute occlusion of the remaining renal artery. Pretreatment with chlorpromazine resulted in a significant attenuation in the rise in postischemic serum creatinine. Hypothermia of the kidney during ischemia provided an additional protective effect. Electron microscopic study of the proximal convoluted tubule demonstrated that the structural damage was less severe in chlorpromazine-treated rats and virtually complete preservation of a normal ultrastructure was observed when hypothermia was added.     

Altered acetylcholine and norepinephrine concentrations in diabetic rat hearts. Role of parasympathetic nervous system in diabetic cardiomyopathy

The concentrations of acetylcholine (ACh) as a parasympathetic marker and norepinephrine (NE) as a sympathetic marker were investigated in the hearts of rats 2, 4, and 8 wk after the induction of diabetes by an injection of streptozocin (STZ; 65 mg/kg i.v.). ACh and NE were measured by high-performance liquid chromatography with electrochemical detection. Diabetic rats showed low body weight and heart weight at 2, 4, and 8 wk and higher heart-to-body weight ratio and bradycardia at 8 wk, almost all of which were normalized after insulin treatment. Myocardial ACh and NE concentrations in the diabetic rats at 2 and 4 wk were not significantly different from those in age-matched control rats. However, ACh and NE concentrations in the diabetic rats at 8 wk significantly increased compared with the control rats. Diabetic rats at 8 wk also had increased myocardial choline concentration and choline acetyltransferase activity and decreased acetylcholinesterase activity. Insulin treatment normalized all of these changes in the diabetic rats. Thus, in STZ-induced diabetes (STZ-D), the concentrations of both cholinergic and noradrenergic neurotransmitters in the myocardium increased. The results of this study confirm a previously reported increase in sympathetic activity to the heart and also indicate that there is an increase in the synthesis and a decrease in the metabolism of ACh in STZ-D and that adequate insulin treatment normalizes these changes.     

Overexpression of metallothionein reduces diabetic cardiomyopathy.

Many diabetic patients suffer from cardiomyopathy, even in the absence of vascular disease. This diabetic cardiomyopathy predisposes patients to heart failure and mortality from myocardial infarction. Evidence from animal models suggests that reactive oxygen species play an important role in the development of diabetic cardiomyopathy. Our laboratory previously developed a transgenic mouse model with targeted overexpression of the antioxidant protein metallothionein (MT) in the heart. In this study we used MT-transgenic mice to test whether an antioxidant protein can reduce cardiomyopathy in the OVE26 transgenic model of diabetes. OVE26 diabetic mice exhibited cardiomyopathy characterized by significantly altered mRNA expression, clear morphological abnormalities, and reduced contractility under ischemic conditions. Diabetic hearts appeared to be under oxidative stress because they had significantly elevated oxidized glutathione (GSSG). Diabetic mice with elevated cardiac MT (called OVE26MT mice) were obtained by crossing OVE26 transgenic mice with MT transgenic mice. Hyperglycemia in OVE26MT mice was indistinguishable from hyperglycemia in OVE26 mice. Despite this, the MT transgene significantly reduced cardiomyopathy in diabetic mice: OVE26MT hearts showed more normal levels of mRNA and GSSG. Typically, OVE26MT hearts were found to be morphologically normal, and elevated MT improved the impaired ischemic contractility seen in diabetic hearts. These results demonstrate that cardiomyocyte-specific expression of an antioxidant protein reduces damage to the diabetic heart.     

Interaction of halogenated anesthetics with alpha- and beta-adrenoceptor stimulations in diabetic rat myocardium

BACKGROUND: Halogenated anesthetics potentiate the positive inotropic effects of alpha- and beta-adrenoceptor stimulations. Although diabetes mellitus induces significant myocardial abnormalities, the interaction of halogenated anesthetics and adrenoceptor stimulation in diabetic myocardium remains unknown. METHODS: Left ventricular papillary muscles were provided from healthy and streptozotocin-induced diabetic rats. Effects of 1 minimum alveolar concentration halothane, isoflurane, and sevoflurane on the inotropic and lusitropic responses of alpha (phenylephrine)- and beta (isoproterenol)-adrenoceptor stimulations were studied at 29 degrees C with 12 pulses/min. Data shown are mean percentage of baseline active force +/- SD. RESULTS: Phenylephrine induced comparable positive inotropic effects in healthy and diabetic rats (143 +/- 8 vs. 136 +/- 18%; not significant), but the potentiation by halogenated anesthetics was abolished in the diabetic rats (121 +/- 20, 130 +/- 20, and 123 +/- 20% for halothane, isoflurane, and sevoflurane, respectively; not significant). In diabetic rats, the positive inotropic effect of isoproterenol was markedly diminished (109 +/- 9 vs. 190 +/- 18%; P < 0.05), but its potentiation was preserved with isoflurane (148 +/- 21%; P < 0.05) and sevoflurane (161 +/- 40%; P < 0.05) but not with halothane (126 +/- 16%; not significant). Halothane induced a deleterious effect on the sarcoplasmic reticulum, as shown by its impairment in the lusitropic effect of isoproterenol, compared with isoflurane and sevoflurane. CONCLUSION: Potentiation of the positive inotropic effect of alpha-adrenoceptor stimulation by halogenated anesthetics is abolished in diabetic rats. In contrast, potentiation of beta-adrenoceptor stimulation is preserved with isoflurane and sevoflurane but not with halothane, probably because of its deleterious effects on sarcoplasmic reticulum.     

Amiodarone pulmonary toxicity: functional and ultrastructural evaluation.

Pulmonary function, chest radiographic appearances, and the cellular composition of bronchoalveolar lavage fluid were assessed in 13 patients who were receiving amiodarone treatment. Eight of the patients had developed clinical and radiological evidence of lung disease and five were symptom free. The proportions of lymphocytes (mean 8.6 (SD 6.9)) and neutrophils (mean 3.4 (3.3)) obtained by bronchoalveolar lavage were similar in patients with and without lung complications. Electron microscopic examination of alveolar macrophages showed intralysosomal inclusion bodies in all subjects, regardless of clinical state. There was no significant difference in the mean number of inclusion bodies per macrophage transection between those with and those without lung disease. The differential cell count in bronchoalveolar lavage fluid and the presence of macrophage inclusion bodies were therefore not useful as markers of disease activity. Among those who developed clinical and radiological evidence of lung disease, the cumulative drug dose per kilogram of body weight and the duration of treatment (mean 16.5 (SD 9.0) months) were significantly correlated with the degree of lung restriction as measured by total lung capacity and forced vital capacity. It is concluded that, while the severity of the restrictive pulmonary defect that is induced by amiodarone is largely dose related, the development of lung toxicity is to some extent idiosyncratic.     

Left ventricular reconstruction benefits patients with ischemic cardiomyopathy and non-viable myocardium.

OBJECTIVE: There are subsets of patients with ischemic cardiomyopathy for whom the optimal treatment strategies are not clear. The objective of this study was to delineate the relationship between clinical outcomes and surgical procedure in patients who were treated either with a coronary artery bypass graft or with a coronary artery bypass graft and additional ventricular restoration. METHODS: The study population comprised 137 consecutive patients with anterior myocardial infarction. All patients had an ejection fraction <50% and left ventricle end-systolic volume index >80 ml/m(2). The patients were divided into a viable and a non-viable group according to anterior myocardium viability, which was determined by a thallium-201 test. The viable group underwent a revascularization and was randomized into two groups for additional ventricular reconstruction. Group 1a comprised 35 patients with viable anterior wall who underwent surgical revascularization. Group 1b comprised 39 patients with viable anterior wall who underwent surgical revascularization and ventricular restoration. Group 2 comprised 69 patients with non-viable anterior wall who underwent revascularization and ventricular reconstruction. The preoperative and postoperative ejection fractions, end-systolic volume, mitral regurgitation, mortality, and heart failure symptoms were compared among groups. RESULTS: Complete 2-year follow-up was achieved in 127 (92.7%) patients. Ejection fraction improved in all groups compared with preoperative values and it was greater in group 1b than in group 1a (p<0.001) at 2 years. There were no postoperative deaths in group 1a, one in group 1b, and two in group 2. After 2 years, group 1b was significantly smaller than group 1a (p<0.01) in relation to mitral regurgitation of grades 1 to 2+. End-systolic volume was significantly smaller in group 1b than in group 1a (p<0.001), it was smaller in group 1a than in group 2 (p<0.001), and it was smaller in group 1b than in group 2 (p<0.001). Heart failure class (NYHA) was reduced in all groups and events were significantly smaller in patients with end-systolic volume lesser than 120 ml/m(2) (p<0.05). CONCLUSION: We have demonstrated that the short-term and mid-term outcomes of coronary artery surgery alone in patients with a large left ventricle are inferior to coronary artery surgery plus ventricular restoration.     

Regeneration of the femoral artery following experimental end-to-end anastomosis in the rat. An ultrastructural follow-up

An experimental end-to-end anastomosis of the common femoral artery was performed in 45 adult Wistar rats. The vessel wall condition was assessed at 10 min, 3 days, 1, 2, 4, and 8 weeks following surgery by scanning and transmission electron microscopy. The microsurgical procedure inflicts extensive trauma in all layers of the arterial wall, with a desendothelialization largely surpassing the surgical field. Reendothelialization was completed at 2 weeks. Apparently, and as compared to less damaging vessel wall manipulations, such deep medial and wide endothelial lesions do not retard vessel wall healing to normal functionality. Moreover, the formation of primary atherogenic structures by smooth muscle cell migration to the intima seems restricted.     

Protection of ischemic myocardium by exogenous phosphocreatine. II. Clinical, ultrastructural, and biochemical evaluations

In valve replacement operations on 78 patients with acquired heart disease, the efficiency of phosphocreatine in intraoperative protection of ischemic myocardium was evaluated by clinical, morphologic, and biochemical methods. Phosphocreatine (8 to 10 mmol/L) in a blood cardioplegic solution was used in operations on 41 patients; in the control group (37 patients) standard blood cardioplegia was used. In the group with phosphocreatine treatment we observed more rapid recovery of hemodynamics after release of the aortic cross-clamp, a decreased frequency of fibrillation, and more frequent restoration of sinus rhythm even if there were sinus rhythm disturbances before aortic cross-clamping. Analysis of the biopsy samples taken from the right ventricle showed protection of the sarcolemma against ischemic damage afforded by phosphocreatine and complete preservation of high-energy phosphates. The results obtained confirm the conclusion made by Robinson, Braimbridge, and Hearse (J Thorac Cardiovasc Surg 1984; 87:190-200) that phosphocreatine is an effective additional cardioprotective agent when used in cardioplegic solutions.     

The spectrum of olfactory neural tumors. A light-microscopic immunohistochemical and ultrastructural analysis

Twenty-eight malignant olfactory neural tumors representative of the histologic spectrum commonly designated as olfactory neuroblastoma were subdivided into two groups: Group I closely resembling classical neuroblastoma (20 cases), and Group II exhibiting neuroendocrine features (eight cases). Immunohistochemically, the tumors were analyzed by using antibodies to keratin, neurofilament protein, S-100, and neuron specific enolase. Neuron specific enolase was the most consistently positive in both groups. Single S-100 positive cells, within or at the edges of tumor nests, often corresponded ultrastructurally to Schwann cells at the tumor-stroma interface. Keratin and neurofilament proteins were expressed singly or together by a small number of cases in both groups. All 11 tumors examined ultrastructurally exhibited neuronal processes containing dense-core granules. The results indicate the following: (a) the reliable diagnostic utility of electron microscopy; (b) the frequent occurrence of Schwann cells in these tumors despite their inconspicuousness by light microscopy; and (c) the unexpected expression of keratin by tumors in both groups. The single or coexpression of keratin-neurofilament protein may define a subset of these tumors for which the clinical significance is presently unclear.     

Biomechanical analysis of experimental spinal cord injury and functional loss

Spinal cord injury was studied using a drop-weight technique in a rat model. A constant weight of 10 g was dropped from 2.5, 5.0 and 17.5 cm heights. The trauma delivered was quantified by biomechanical parameters of weight drop height, maximum force, and impulse, while the functional deficit produced by injury was quantified in terms of the maximum inclined plane score, hindlimb motor score, and combined behavioral score. Highly significant (P = 0.0001) relationships were found within the biomechanical parameters of trauma as well as between the trauma and the functional parameters at 4 weeks after injury. The impulse was found to be the best predictor of the functional loss (r = 0.79, P = 0.0001).     

Clinical and ultrastructural analysis of failures in chemonucleolysis.

A series of 12 patients with failed chemonucleolysis were analyzed to determine the various causes of such failures. The ultrastructural findings observed in laminectomy specimens did not account for the failure in the enzyme treatment. The findings suggested deficient cellular nutrition secondary to enzymatic histolysis of the nucleus pulposus.     

Human islet xenograft survival in diabetic rats. A functional and immunohistochemical study

Prolonged survival of human islet xenografts under the kidney capsule of diabetic rats was achieved. Human islet xenograft survival time for the nonimmunosuppressed and single-dose antithymocyte serum-treated rats were 3.7 +/- 0.33 days (mean +/- SE, n = 6) and 4.2 +/- 0.63 (n = 4), respectively. In the recipients given 5 doses of ATS after islet transplantation, the graft survival time was significantly prolonged to 18.2 +/- 1.9 days (n = 6). An intravenous glucose tolerance test was performed on 3 recipients with a functional graft 12 days after xenotransplantation. The mean K rate was 1.44 +/- 0.43 (n = 3) compared with that of 2.1 +/- 0.14 (n = 5) found in normal control rats. Human C-peptide was present in the rat recipients following islet transplantation. In addition all 3 recipients showed significant basal human C-peptide values posttransplant and achieved levels of above 2.4 ng/ml during IVGTT. Morphologic and immunohistochemical examination of the islet grafts show that in recipients without immunosuppression or with a single dose of ATS, there was marked degree of fibrosis with little endocrine tissue left in the graft area by day 5. In contrast, the xenograft from recipients treated with 5 doses of ATS still contained well-preserved islet tissue with many insulin and glucagon containing cells on the day of graft removal when blood glucose had returned to the hyperglycemic level. Infiltration of the graft area with lymphoid cells (OX1+, OX8+, and W3/25+) was prominent, but they were not detected within the islets. Staining with monoclonal antibody clone L243 did not detect any expression of human class II antigen on the human pancreatic endocrine cells undergoing rejection by the host. This study has shown that with adequate immunosuppression human islet xenograft can normalize the blood glucose with prolonged survival time in diabetic rat recipients. The discordant xenotransplantation model used in this study would be useful for future xenotransplantation studies.     

Pseudointima formation in woven and knitted dacron grafts. A comparative ultrastructural analysis

Twelve specially designed crimped aortobifurcated grafts (aorta, woven 200 porosity; right limb, woven 500 porosity; left limb, knitted 1200 porosity) were implanted into mongrel dogs that were sacrificed at two, six, eight and twelve-month intervals. Pseudointima from the mid portion of each type of graft was studied with light (LM) and transmission electron microscopy (TEM). An ocular micrometer was used to determine the thickness of the pseudointima. A trilaminar cytoarchitecture resembling the wall of a medium-sized artery existed in the pseudointima. This was characterized by an inner cellular layer, outer fibrocollagenous layer, and multi-interlamination of cells and glycosaminoglycan in the middle layer. The cellular inner layer was thickest in the knitted and thinnest in the woven 200 porosity. A zone of acellularity separated the luminal cells from the underlying myoblasts and myofibroblasts. Specificity of varying types of cell distribution occurred in the pseudointima. Myoblasts and myofibroblasts were located near the lumen while fibroblasts and mesenchymoid cells were situated near the graft. The luminal cells had the combined features of endothelial cells and myofibroblasts. Collagen and glycosaminoglycans were the dominant extracellular matrix. Despite the difference of fluid permeability in high porosity woven and knitted dacron grafts, the pseudointima was well formed and revealed no striking general cytoarchitectural difference between these two types of grafts.     

Mucosal alterations in pelvic ileal reservoirs. A histological and ultrastructural evaluation in an experimental model

To study mucosal changes in ileal pouches, three different types of pelvic ileal reservoir (2-loop, 3-loop and 4-loop), interposed in place of resected rectum, were constructed in 12 piglets, 4 animals in each group (n = 12). After a 6 week interval the animals were sacrificed and the reservoirs removed and processed for light and scanning electron microscopy (SEM). Both acute and chronic inflammatory changes were present but not prominent in the pouch mucosa. With the exception of mucosal ulcers both the histological and SEM-findings greatly resembled the atrophic, flat mucosa seen in ileal coeliac disease. Macroscopic findings did not correlate with histological findings. Colonization by bacteria and bacterial overgrowth were noticed in every pouch suggesting a possible link between bacterial invasion and pouch ileitis. No correlation was found, however, between the amount of intraluminal bacteria and the severity of the pouchitis.