Multidimensional Analysis of the Adult Human Heart in Health and Disease using Hierarchical Phase-Contrast Tomography (HiP-CT).

Cardiovascular diseases (CVDs) are a leading cause of death worldwide. Current clinical imaging modalities provide resolution adequate for diagnosis but are unable to provide detail of structural changes in the heart, across length-scales, necessary for understanding underlying pathophysiology of disease. Hierarchical Phase-Contrast Tomography (HiP-CT), using new (4th) generation synchrotron sources, potentially overcomes this limitation, allowing micron resolution imaging of intact adult organs with unprecedented detail. In this proof of principle study (n=2), we show the utility of HiP-CT to image whole adult human hearts ex-vivo: one 'control' without known cardiac disease and one with multiple known cardiopulmonary pathologies. The resulting multiscale imaging was able to demonstrate exemplars of anatomy in each cardiac segment along with novel findings in the cardiac conduction system, from gross (20 um/voxel) to cellular scale (2.2 um/voxel), non-destructively, thereby bridging the gap between macroscopic and microscopic investigations. We propose that the technique represents a significant step in virtual autopsy methods for studying structural heart disease, facilitating research into abnormalities across scales and age-groups. It opens up possibilities for understanding and treating disease; and provides a cardiac 'blueprint' with potential for in-silico simulation, device design, virtual surgical training, and bioengineered heart in the future.

Growth factor staining patterns in the pig retina following retinal laser photocoagulation.

AIM: To identify changes in growth factor expression in miniature pig retinas following retinal laser photocoagulation. METHODS: Pigs were sacrificed at different times (15 minutes to 42 days) post-laser and the retinas were immunolabelled for basic fibroblast growth factor, insulin-like growth factor I, transforming growth factor beta, epidermal growth factor, transforming growth factor alpha, platelet derived growth factor, vascular endothelial growth factor, and epidermal growth factor receptor. Total mRNA levels were also determined. RESULTS: With the exception of vascular endothelial growth factor, immunoreactivity for all other growth factors studied and epidermal growth factor receptor was observed throughout normal non-lasered control retina, generally being high in the retinal pigment epithelium and low in the neural retina. Changes in growth factor expression following laser photocoagulation were observed only in burn areas and changes were mainly confined to the retinal pigment epithelium and outer nuclear layer. The immunoreactivity within retinal pigment epithelial cells in burn areas was either absent or decreased following laser treatment but returned to normal by 21 days. The immunoreactivity was increased within the outer nuclear layer of burn areas during the healing process but returned to normal by 42 days. Vascular endothelial growth factor immunoreactivity was weak/absent in the normal retina and remained unchanged following laser photocoagulation. Change of total mRNA levels in burn areas during time post-laser was confined to retinal pigment epithelial cells, being low immediately following photocoagulation and returning to normal by 42 days. CONCLUSIONS: These results demonstrate a temporal alteration in growth factor expression and transcriptional activity in the retina following laser photocoagulation.

Venous thrombotic complications of pregnancy.

A total 30,040 pregnancies were reviewed at one institution over 5 years to determine the incidence of venous thrombotic complications. Thirty-one patients experienced such complications related to pregnancy (incidence 0.1%); 13 had deep venous thrombosis and 14 had superficial venous thrombophlebitis diagnosed by duplex ultrasound. Four had pelvic vein thrombophlebitis diagnosed by computed tomography scan; three patients (one from each group) sustained a non-fatal pulmonary embolus. Of those with deep venous thrombosis, 10 (77%) were left-sided, and three (23%) were right-sided. Three had a prior history of deep venous thrombosis and one of pulmonary embolism. Of those with superficial venous thrombophlebitis, seven (50%) were left-sided, six (43%) were right-sided, and one (7%) was bilateral. Most with deep venous thrombosis presented later in pregnancy; three in the first trimester, two in the second, three in the third, and five early postpartum. Most (10/14) with superficial venous thrombophlebitis presented within 48 hours of delivery. Distribution of thrombi in those with deep venous thrombosis was compared with 643 non-pregnant women with a similar condition. A pattern of proximal involvement on the left was found, with left common femoral vein (54% versus 28%, P = 0.03) and superficial femoral vein (62% versus 26%, P = 0.006) more often involved in pregnant patients. The average number of vein segments involved was greater on the left than the right (5.3 versus 3.7). Symptoms of chronic venous insufficiency developed in three with deep venous thrombosis (25%) and in three with superficial venous thrombophlebitis (27%). None had recurrence of deep venous thrombosis. It is concluded that venous thrombotic complications associated with pregnancy are not necessarily benign, with the risk of pulmonary embolism and chronic venous insufficiency not limited to patients with deep venous thrombosis only.

The effect of scatter laser photocoagulation on intravitreal levels of growth factors in the miniature pig.

PURPOSE: To characterise changes in intravitreal growth factor profiles following retinal photocoagulation in the miniature pig. METHODS: Miniature pig eyes underwent scatter photocoagulation by either diode infrared or emerald green laser. Animals were sacrificed at various times (up to 42 days) post-laser. The eyes were than removed and vitreous samples analysed for basic fibroblast growth factor, insulin-like growth factor-I and epidermal growth factor by radioimmunoassay, transforming growth factor-beta 2 by ELISA and insulin-like growth factor binding proteins using Western ligand blotting. RESULTS: Vitreous transforming growth factor-beta 2 levels were decreased at 1 h post diode laser and at 4 and 7 days post emerald laser but returned to normal by 21 and 42 days respectively. Vitreous insulin-like growth factor-I levels increased at 4 and 7 days post diode and emerald laser respectively but returned to normal by 21 days. Insulin-like growth factor Western ligand blotting demonstrated that a 34 kDa insulin-like growth factor binding protein was predominant in the pig vitreous; the levels of this binding protein followed an identical trend to those observed for insulin-like growth factor-I. No changes in vitreous levels of either basic fibroblast growth factor or epidermal growth factor were observed following laser treatment. CONCLUSIONS: Our results demonstrate a significant shift in the balance of intravitreal growth factors following retinal laser photocoagulation. Such changes may be pertinent to the regression of preretinal new vessels after laser photocoagulation.

Nociceptive somatic nerve stimulation and skeletal muscle injury modify systemic hemodynamics and oxygen transport and utilization after resuscitation from hemorrhage.

OBJECTIVE: To examine if either nociceptive somatic nerve stimulation or skeletal muscle injury modified systemic hemodynamics and oxygen transport and utilization after resuscitation from hemorrhage in anesthetized pigs. DESIGN: Prospective, randomized, controlled laboratory study. SETTING: Animal laboratory. SUBJECTS: Twenty isoflurane-anesthetized and mechanically ventilated large white pigs. INTERVENTIONS: Three groups of animals were instrumented with femoral arterial and thermodilution pulmonary artery catheters. One group of animals had bilateral brachial nerve electric stimulation before hemorrhage (brachial nerve stimulation + hemorrhage, n = 7). The second group of animals had bilateral hindlimbs skeletal muscle injury induced by firing a captive-bolt handgun with standard charges before hemorrhage (skeletal muscle injury + hemorrhage, n = 6). The third group had neither insult before hemorrhage (control, n = 7). Controlled bleeding was initiated to reduce the cardiac index and systemic oxygen delivery (Do2) by 50% in all animals. Animals were then left for 30 mins before resuscitation. All animals were resuscitated with 4.5% human serum albumin at 45 mL/kg and observed for 2 hrs. MEASUREMENTS AND MAIN RESULTS: Plasma volume, systemic hemodynamics, and oxygen transport variables were measured and calculated after resuscitation. Similar increases of plasma volume and supranormal cardiac index were observed in all groups immediately after resuscitation. The branchial nerve stimulation and hemorrhage group maintained higher heart rate, cardiac index, Do2, and oxygen consumption (Vo2) than the hemorrhage group. In contrast, the skeletal muscle injury and hemorrhage group had lower systemic mean arterial pressure and vascular resistance, and a tendency for decrease in Vo2, than the hemorrhage group, although heart rate, cardiac index, and Do2 were similar in both groups. Hemorrhage increased the arterial plasma lactate concentration, which was later normalized in all groups 60 mins after resuscitation. CONCLUSIONS: Neither nociceptive brachial nerve stimulation nor skeletal muscle injury attenuated the increase in plasma volume, cardiac index, or the repayment of systemic oxygen debt after resuscitation from hemorrhage. Brachial nerve stimulation was associated with augmented cardiac index, systemic Do2, and increased Vo2 requirements related to increased sympathetic nervous system activation. Skeletal muscle injury produced early systemic arterial hypotension and vasodilation, and a decrease in Vo2 that was suggestive of pathologic supply dependency on systemic Do2.

Latissimus dorsi muscle blood flow during synchronized contraction: implications for cardiomyoplasty.

BACKGROUND: Damage in latissimus dorsi muscle flaps has been reported after clinical and experimental cardiomyoplasty, and an ischemic origin has been suggested. METHOD: In situ, preconditioned latissimus dorsi muscles in 5 sheep were stimulated in either 1:1 (muscle: heart) or 1:2 synchrony with the systolic phase of the cardiac cycle, using a burst duration of either 21% or 35% of the cycle. Thoracodorsal artery blood flow and thoracodorsal venous lactate concentrations were measured before and immediately after a 3-minute period of stimulation. RESULTS: The exercise-induced augmentation of thoracodorsal artery blood flow was significantly (p < 0.05) less with a 1:2 regimen than a 1:1 regimen, for both a 21% (88%; 95% confidence interval [CI], 55.6% to 127.3% versus 138.9%; CI, 97.6% to 188.8%) and 35% burst duration (123.2%; CI, 84.7% to 169.9% versus 167.0; CI, 120.8% to 222.6%). After cessation of stimulation, reactive hyperaemia was observed in 3 of 5 animals with 1:1 21% burst stimulation, and in 5 of 5 animals with a 35% burst duration, but was not seen after 1:2 regimens. A significant (p < 0.01) increase in thoracodorsal venous lactate levels was present after 1:1 35% burst stimulation (34.9%; CI, 9.9% to 65.6%), but lactate levels tended to fall when a 1:2 ratio was used (15.9%; CI, -3.2% to 31.5%; p < 0.1). CONCLUSIONS: One-to-one stimulation regimens may be detrimental to latissimus dorsi blood flow, and an adaptive, rather than fixed, burst duration may be preferable. These findings have important implications for the cardiomyoplasty procedure.

The flank approach to the porcine upper urinary tract: safe and reliable.

An extra-peritoneal flank approach was used to gain access to the left pelviureteric junction of 8 young pigs. The operative procedure was simple and took 15 min to complete. There were no serious complications, analgesia requirements were negligible and all animals re-established on feeds within 12 h post-operatively. The operative procedure is described; due to the above considerations the flank approach to the porcine kidney can be recommended.

A comparison of the brain weights of normal, untreated hydrocephalic and treated hydrocephalic rat pups.

The dry brain weights of normal, untreated and treated hydrocephalic pups of Texas, Lewis and Texas/Lewis cross rats aged from 2 to 60 days were compared using the technique of generalised linear modelling. It was found that with increasing age there is a steadily progressive increase in the weight of the brains of the normal animals. The brains of the untreated hydrocephalics showed a normal increase in weight up to about the 25th day but after that whilst the weight continued to increase, it was significantly less than normal. On the other hand the weight gain of the brains of the treated hydrocephalic pups resembled that of the normals and steadily increase in weight throughout the period of observation.

Does the asymptomatic limb harbor deep venous thrombosis?

Deep venous thrombosis (DVT) is a great masquerader that cannot be reliably predicted by a patient's symptoms, history, or risk factors. Bilateral lower extremity duplex ultrasonography scans were made of 2,511 patients and analyzed to identify, if possible, a population in which a unilateral study would be appropriate. A total of 1,086 (43%) patients were found to have deep venous thrombosis--742 (30%) unilateral and 344 (14%) bilateral. Of the patients with DVT for whom side-of-symptom information was recorded, 64% had symptoms referable to the involved extremity and 36% had symptoms referable to the contralateral extremity. Of the 362 patients who had asymptomatic lower extremities, 128 (35%) had DVT. Moreover, clots were found in asymptomatic limbs in an additional 263 patients whose contralateral limb was symptomatic. Logistic regression analysis did not reveal combinations of symptoms and risk factors that could predict DVT. If DVT is suspected, the patient should undergo bilateral lower extremity duplex scanning.

A comparison of the effects of skeletal muscle injury and somatic afferent nerve stimulation on the response to hemorrhage in anesthetized pigs.

The effect of skeletal muscle injury (SMI) on cardiovascular and O2 transport responses to hemorrhage (HS) were examined in anesthetized pigs. Bilateral hindlimb muscle was injured 75 minutes before HS was started at a rate of 0.75 mL/min.kg until a total of 30 mL/kg (40% estimated total blood volume) had been removed. The reductions in cardiac index (CI), left ventricular stroke work, and oxygen delivery (Do2) and the increase in plasma lactate concentration following HS were exacerbated by SMI such that although oxygen consumption was maintained after HS it fell after SMI + HS. The deleterious effect of SMI on the response to HS was greater than that recorded previously following somatic brachial nerve stimulation (BNS). Thus, in order to achieve a given reduction in CI and Do2 or a rise in Shock Index (heart rate divided by systolic blood pressure) to approximately 3, a blood loss of 40% was needed after HS; this was reduced to 36% by the addition of BNS, whereas a loss of only 29% was needed when SMI was introduced. The mechanism of the deleterious effect of SMI is unclear although a change in the distribution of regional blood flow and a rise in the critical oxygen delivery may be implicated.

Experience with physiologic amputation using the CryoCare Extremity Stabilization System (CESS).

Five years of experience gained with the CryoCare Extremity Stabilization System (CESS) were evaluated in this study. Twenty-one patients underwent freezing amputation. Five patients died before undergoing surgical amputation. Symptomatic relief, control of odor, decreased demand on nursing staff, and appreciation of the family make this approach valuable even when long-term survival is not anticipated. Ten patients who underwent freezing amputation subsequently underwent surgical amputation and were discharged. Six patients underwent freezing and surgical amputation but died prior to discharge. The patients selected for the freezer application were deemed to be prohibitive operative risks because they were experiencing systemic toxicity from their ischemic limb and underlying diseases. Six patients demonstrated myoglobinuria prior to freezing which cleared with CESS. The physiologic amputation allowed stabilization of medical problems including cardiac arrhythmias, congestive heart failure, sepsis, renal failure, diabetes, and respiratory failure. Freezing of an ischemic extremity allows delay in amputation enabling physicians to achieve maximal medical stabilization. It permits symptomatic relief in patients whose long-term survival is not anticipated. Physiologic freezing amputation should be included in the repertoire of all surgeons.

Upper extremity venous duplex imaging.

Over 500 upper extremities have been imaged with the Duplex Scanner at the John J. Cranley Vascular laboratory. Venous anatomy and imaging techniques are presented. Reference points and zones for localizing abnormal findings are demonstrated. Abnormal venous pathology including case presentations of acute radial, basilic, cephalic, and axillary vein thrombosis are presented. Complications of subclavian and internal jugular vein catheterization are demonstrated. Soft tissue pathology including edema, lymphadenopathy, hematomas, cysts, and abscesses are visualized and differentiated. Duplex use for evaluation of aneurysm, pseudo aneurysm and graft surveillance has been increasing in frequency. Examples are demonstrated.

Lower extremity calf thrombosis: to treat or not to treat?

Seventy-five patients with isolated calf vein thrombi were prospectively monitored with sequential duplex scans at 3- to 4-day intervals. Twenty-four patients (32%) propagated and 11 of these 24 (46%) into the popliteal or larger veins of the thigh. Sex, age, obesity, trauma, estrogen use, malignancy, varicose veins, smoking, surgery, and activity level were not predictive for proximal propagation. Proximal soleal vein thrombi had the highest incidence in both propagating and non-propagating groups. Thrombus extent and bilateral involvement were not predictive of propagation. Five percent (4 of 75 patients) had highly probable ventilation perfusion scans as their initial indication for duplex scanning. Deep vein thrombosis isolated to the calf is not a benign problem. If anticoagulant therapy is contraindicated, the progress of the thrombus can be followed by duplex scanning.

Measurement of blood flow rates in the lower extremities with use of a nuclear magnetic resonance based instrument.

Direct, noninvasive measurement of pulsatile blood flow to the human extremity is now possible by means of a flow measurement instrument that is based on the principles of nuclear magnetic resonance. The instrument uses a physically independent calibration module as a primary calibration standard. Volumetric calibration of this module indicates that it is precise and accurate over the range of 0 to 100 ml/min. The calibration module is used, in turn, to calibrate an electromagnetic flow sensor that is incorporated into the instrumentation. The calibration module and the electromagnetic sensor were found to be linearly related over the range of 5 to 100 ml/min, with a regression correlation coefficient of 0.996. The calibrated electromagnetic flow sensor is used as a secondary standard for calibration of the nuclear magnetic resonance sensor. Blood flow measurements, obtained by use of this method, agree closely with those obtained by plethysmographic methods. They differ from the plethysmographic results in that magnetic resonance flows will distinguish between the at-rest blood flow in the normal extremity and the flows seen in the extremity (also at rest) with claudication. Based on the results obtained from studying a limited number of limbs with a high degree of ischemia, the method will not distinguish the limb with ischemia from the limb with claudication. Limitations of the method and refinements required to make the method clinically useful are discussed.

Superficial thrombophlebitis diagnosed by duplex scanning.

Since July 1982, this noninvasive vascular laboratory has performed 12,856 lower extermity venous duplex examinations. All cases of acute venous thrombosis have been categorized and entered into a computer data base. One thousand four hundred twelve examinations were positive for acute venous thrombosis. This report analyzes the laboratory's entire experience with superficial thrombophlebitis (SVT). One hundred eighty-six patients were diagnosed by duplex scanning to have SVT. Women outnumbered men 99 to 87. They were slightly older (average age 58.4 +/- 16.2 years) compared with the men (53.8 +/- 14.2 years). Men were more likely to have a complicated course of SVT (40% vs 22%; p less than 0.01). Complications included either radiographically documented pulmonary embolism or deep venous involvement. Fifty-seven (31%) patients had at least one complication of SVT. A series of predisposing factors was analyzed and six factors were associated with an increased risk of complications. They are bilateral SVT (p less than 0.01), age greater than 60 years (p less than 0.01), male sex (p less than 0.01), history of deep venous thrombosis (p less than 0.01), bed rest (p less than 0.02), and presence of infection (p less than 0.02). Location of thrombus within the greater saphenous vein (35%) was most likely to be associated with complications. Isolated varicosities (8%) were least likely to be associated with complications. Duplex scanning identifies a significant number of complications of patients with SVT and should be obtained in cases of saphenous vein involvement or in the presence of associated risk factors.

Comparison of healing of lesions in the calvarium of foetal lambs and young sheep.

This study compares the healing of lesions made in the occipital region of the calvarium of 5 foetal and 4 post-natal lambs. The foetuses, operated on between the 83rd and 91st day of gestation, had a bone flap elevated and the post-natal lambs, aged about 6 weeks, had a craniectomy and replacement of the bone pieces. The foetuses were delivered spontaneously at full-term. All the animals had computed tomographic (CT) scans and, after sacrifice, naked eye examination of the bony lesions. These showed that there was poor ossification of the lesions made in the foetuses compared to those made in the post-natal lambs. These findings need to be taken into account when intra-uterine correction of cranio-facial deformities are being contemplated.

Analysis of 1084 consecutive lower extremities involved with acute venous thrombosis diagnosed by duplex scanning.

A retrospective analysis of 8658 consecutive lower extremity venous duplex scans performed between the years 1982 to 1988 revealed 953 patients with involvement of 1084 extremities with acute deep or superficial thrombi. Records of patients with acute thrombi were then evaluated for the incidence, location, and patterns of distribution. There were 485 women (50.9%) and 468 men (49.1%), with a mean age of 62.9 +/- 16.7 years and 58.8 +/- 15.2 years, respectively. There were 371 right-sided thrombi (180 women and 191 men), 451 left-sided thrombi (235 women and 216 men), and 131 (70 women and 61 men) patients with thrombi in both lower extremities. Women were found to be uniformly older, and the left leg was found to be involved more frequently (p less than 0.05). The overall distribution of the 3169 veins involved with acute thrombi in decreasing order were: popliteal, 16.1%; superficial femoral, 15.0%; posterior tibial, 13.4%; common femoral, 13.2%; greater saphenous, 9.9%; soleal, 9.1%; peroneal, 7.2%; deep femoral, 6.6%; lesser saphenous, 5.7%; anterior tibial, 2.0%; varicosities, 1.6%; and perforating, 0.3%. A different rank order was found in analysis of single thrombus patterns as follows: greater saphenous, 27.5%; soleal, 20.1%; lesser saphenous, 13.4%; varicosities, 8.8%; popliteal, 8.1%; posterior tibial, 9.1%; common femoral, 3.5%; superficial femoral, 4.9%; peroneal, 2.8%; deep femoral, 1.0%; anterior tibial, 0.3%; and perforating, 0.3%. In patients with multiple and bilateral thrombi there was a large number of unique patterns of thrombosis. Locations, patterns, and frequency of acute venous thrombi vary with age, sex, and leg involved. Patterns and statistical analyses of pertinent observations were performed.