Pulsed electromagnetic fields as adjuvant therapy in bone healing and peri-implant bone formation: an experimental study in rats.

The objective of this study was to determine whether short exposure to pulsed electromagnetic fields (PEMF) accelerates bone repair and peri-implant bone formation in a rat tibial model at different times. Sixty Wistar rats were employed. Sterile custom fabricated commercially pure cylinder threaded titanium implants were placed in the right tibial crest, and an osteotomy was performed in the left tibial crest of each animal. Thirty rats were treated with PEMF (72 mT 50Hz), twice a day in sessions of 30 minutes each, and 30 rats of the control group were sham-treated. Rats were sacrificed at 5, 10 and 20 days postsurgery (n = 10 per group). Tibias were fixed in formaldehyde and decalcified, embedded in paraffin, and stained with hematoxylin-eosin (half samples of left tibias), or they were included in methylmethacrylate, grinded and polished (right tibias and half samples of left tibias). Bone healing was evaluated by image analysis in terms of ossification area, and perimeter and diameter of the lesion. Peri-implant ossification was assessed in terms of ossification percentage. At day 10 the area of ossification index was higher in the PEMF group than in the control group (p = 0.012). At day 20 the osteotomies of the PEMF group were almost completely remodeled. The ossification percentage was higher in the PEMF group (p = 0.018). In conclusion, short daily electromagnetic stimulation appears to be a promising treatment for acceleration of both bone-healing and peri-implant bone formation.

Relationship between oxidative stress, lipid peroxidation, and ultrastructural damage in patients with coronary artery disease undergoing cardioplegic arrest/reperfusion.

OBJECTIVE: In animal models, formation of oxidants during postischemic reperfusion may exert deleterious effects ("oxidative stress"). Cardioplegic arrest/reperfusion during cardiac surgery might similarly induce oxidative stress. However, the phenomenon has not been precisely characterized in patients, and therefore the role of antioxidant therapy at cardiac surgery is a matter of debate. Thus, we wanted to ascertain whether the relationship between oxidant formation and development of myocardial injury also translates to the situation of patients subjected to cardioplegic arrest. METHODS: In 24 patients undergoing coronary artery bypass, trans-cardiac blood samples and myocardial biopsies were taken before cardioplegic arrest and again following reperfusion. RESULTS: Cardiac glutathione release (marker of oxidant production) was negligible at baseline (0.02+/-0.04 micromol/L), but it increased 15 min into reperfusion (1.10+/-0.40 micromol/L; p<0.05); concomitantly, myocardial concentration of the antioxidant ubiquinol decreased from 144.5+/-52.0 to 97.6+/-82.0 nmol/g (p<0.05). Although these changes document cardiac exposure to oxidants, they were not accompanied by evidence of injury. Neither coronary sinus blood nor cardiac biopsies showed increased lipid peroxide concentrations. Furthermore, electron microscopy showed no major ultrastructural alterations. Finally, full recovery of left ventricular systolic and diastolic function was observed. CONCLUSIONS: Careful investigation reveals that while oxidant production does occur during cardiac surgery in patients with chronic ischemic heart disease, cardiac oxidative stress may not progress through membrane damage and irreversible injury.

Mitochondrial oxidative and structural damage in ischemia-reperfusion in human myocardium. Current knowledge and future directions.

The sequence of events in heart ischemia-reperfusion has been clearly documented in experimental animal models but not in cardiac surgery patients. The evidence in human studies had not been gathered in a systematic and comprehensive fashion, so as to provide an encompassing picture of the phenomenon. This limits our ability to devise appropriate strategies for optimal perioperative myocardial protection. We present here a review or our experience in myocardial ischemia-reperfusion in a historical perspective. From our previous studies we conclude that, although several issues still remain unsolved, there is no doubt that oxygen-free radicals are important contributors to myocardial injury during the reperfusion period of coronary artery bypass surgery. Yet, in spite of this wealth of information, both clinical and experimental, subsequent clinical trials conducted over the last several years with a variety of antioxidant strategies have been largely disappointing. Therefore, the whole paradigm of oxidative stress in cardiac injury needs to be re-evaluated. In this regard, differences between past and current knowledge are discussed, and future directions are traced. We concluded that patients subjected to elective bypass surgery undergo oxidative stress upon reperfusion after cardioplegic arrest; the magnitude of the phenomenon, however, is at present small and may not justify widespread antioxidant therapy.

Ultrastructural evidence of increased tolerance of hibernating myocardium to cardioplegic ischemia-reperfusion injury.

OBJECTIVES: The goal of this study was to investigate the effects of ischemia-reperfusion on myocardial ultrastructure in patients with and without hibernating myocardium. BACKGROUND: It is generally accepted that chronically dysfunctional, hibernating myocardium may remain nonetheless viable for a long time. It has been postulated that hibernating myocytes may survive, despite being subtended by a severe coronary artery stenosis, as they might be less susceptible to ischemic insults. However, whether hibernating myocardium is indeed more resistant to ischemia has never been investigated. METHODS: Myocardial biopsies were taken before cardiac arrest and after reperfusion from the anterior wall of the left ventricle in patients undergoing coronary artery bypass surgery, divided according to presence (n = 7) or absence (n = 7) of hibernating myocardium. Ultrastructural changes were studied by electron microscopy. Because ischemia-reperfusion injury is related to oxidative stress, we also evaluated coronary sinus concentration of the antioxidants alpha-tocopherol, beta-carotene, and ubiquinol, and of lipid peroxidation products pre-ischemia and after reperfusion. RESULTS: Both groups were similar with respect to length of ischemia and changes in the various indexes of oxidative stress. In normally contracting myocardium, ischemia/reperfusion induced moderate overall ultrastructural changes, and marked alterations at the mitochondrial level. In contrast, post-reperfusion biopsies of hibernating myocardium displayed only minor overall ultrastructural changes, and scored significantly better on mitochondrial damage. CONCLUSIONS: Despite similar severity of ischemia/reperfusion, hibernating myocardium showed significantly less ultrastructural evidence of cell injury compared with normally contracting myocardium. These data indicate that human hibernating myocardium is intrinsically more resistant to ischemia/reperfusion injury.

Severe carotid barochemoreceptor involvement in stroke.

BACKGROUND: Physiopathology of barochemoreception is hindered by the scarce information on its morphology in disease. The baroreflex is of major importance for the maintenance of arterial pressure during orthostatic stress. The purpose of this paper was to characterize the morphological alterations of carotid glomus in old patients who died from stroke and suffering obstructive carotid atheromatosis. METHODS: Bilateral carotid segments were obtained at autopsy from 17 patients (51-89 years old). Specimens were stained with hematoxylin and eosin; Azan trichrome, Grimelius silver stain for catecholamine detection, and were immunophenotyped for CD34 and S-100. Similar segments of both carotid arteries of six patients (62-77 years old) who died by accidents were used as controls. RESULTS: The carotid glomus of patients who died from stroke presented atrophy and fibrosis (2.59+/-0.5 vs. 1.17+/-0.39 in the control group; p<0.0001). There was a loss of chief cells and of the argyrophilic staining granules. A focal diminution of glomus vascularization was observed in the areas of atrophy and fibrosis (2.73+/-0.45 vs. 1.5+/-0.52 in the control group; p<0.0001). The arterioles to glomus showed severe fibrointimal proliferation, disruption of internal elastic lamina and luminal narrowing, and luminal thrombi. CONCLUSION: A severe carotid glomic damage does exist in old patients who died from stroke and suffering from carotid atheromatosis, independent from aging, of note, a "culprit" marked narrowing of the corresponding arterioles was observed.

Plasminogen activator inhibitor-1 and transforming growth factor-beta 1 in carotid glomus and autonomic ganglia from spontaneously hypertensive rats.

BACKGROUND: Baroreflex and chemoreflex mechanisms play an important role in the dynamic adjustments of circulation and ventilation during daily life. Recently, we have observed atrophy and marked fibrosis in carotid glomus (CG) from old patients with carotid atherosclerosis who died following stroke. However, a possible limitation to interpretation of the results in that study was the superposition of arterial hypertension, atherosclerosis and aging in the patients. Taking this into account, spontaneously hypertensive rats (SHR) were used in order to study the CG in an experimental model with only hemodynamic stress. OBJECTIVE: To evaluate whether transforming growth factor-beta 1 (TGF-beta 1) and plasminogen activator inhibitor-1 (PAI-1) were involved in the extracellular matrix expansion in CG and autonomic ganglia (AG) in young, male, adult SHR. METHODS: Male SHR (n = 10) and Wistar-Kyoto (WKY) rats (n = 10) were used. Systolic blood pressure (SBP) was measured monthly up to 8 months of age, when the animals were killed; then, CG and AG were excised and processed for light microscopy and immunohistochemistry (TGF-beta 1, PAI-1 and protein S100). RESULTS: SBP was highly correlated (P < 0.01) with CG fibrosis (r = 0.90), AG fibrosis (r = 0.96) and neuron number (r = -0.97). PAI-1 and TGF-beta 1 in CG and AG were significantly increased (P < 0.01) in SHR. CONCLUSION: Severe damage was observed in CG and AG in SHR, which was, in addition, correlated with SBP. These results suggest that permanent high blood pressure produces remarkable lesions in these structures, even when the animals are not old. In view of the fact that CG and AG are of utmost importance in the genesis of cardiocirculatory reflexes, they might be considered as 'target organs' in arterial hypertension.

Expression of c-fos, p53 and PCNA in the unstable atherosclerotic carotid plaque.

BACKGROUND: The process by which a fibrofatty plaque evolves into a fibrotic lesion or into an unstable, lipid-rich plaque is poorly understood. In this study our aim is to deepen the knowledge of the cellular proliferation mechanisms that characterize the initial phases of destabilization of the unstable carotid plaque. METHODS: 32 specimens from carotid endarterectomies were employed to assess by immunohistochemical methods, either in stable (n=10) or unstable (n=22) atherosclerotic plaques, the proliferating cell nuclear antigen (PCNA), the proto-oncogene c-fos, and the oncoprotein p53. RESULTS: 18/32 atherosclerotic plaques (all unstable), showed c-fos immunopositivity (P<0.0001). Ten lesions, three stable and seven unstable, were PCNA+, while 13 cases were positive for p53 (three stable and 10 unstable plaques). When comparing symptomatic vs. asymptomatic patients, the most striking finding was the coincidence between c-fos, PCNA and p53 protein positivity observed only in unstable plaques of seven out of eight patients, all with previous episodes of stroke or transient ischemic attacks. On the other hand, none of the above mentioned positivity was detected in the 24 asymptomatic patients (P<0.0001). CONCLUSIONS: These findings indicate an important role of these biomarkers in vascular biology. A series of molecular pathways of disease development and progression common both to atherosclerosis and cancer, support that the world's two most common diseases are more closely aligned than previously believed.

Histomorphometry of umbilical cord blood vessels in preeclampsia.

The authors hypothesized that preeclampsia may change the phenotype of umbilical cord vessels. Segments of umbilical cords were obtained from 29 pregnant women (20 healthy and 9 with preeclampsia), which were histomorphometrically assessed. Birth weight was 2928 ± 613 g for the control group vs 1749 ± 656 g for the preeclampsia group (P<.0001). A significantly shorter gestational period was noted in the preeclampsia group: 35 weeks vs 39 weeks in the healthy group. Measurements of the outer layer area (116.4 ± 55 µm(2) vs 56.5±25 µm(2) ; P=.0038), the inner layer area (63.1 ± 16 µm(2) vs 28.6±8 µm(2) ; P<.0001), the lumen area (8.4 ± 1 µm(2) vs 3.4±2 µm(2) ; P=.0003), and the wall/lumen ratio (20.3 ± 9 vs 3.1 ± 0.6; P<.0001) of arteries were significantly larger in the preeclampsia umbilical cords. Concerning veins, the wall/lumen ratio was higher in the preeclampsia group. In this study, the umbilical cord in preeclampsia showed significant changes in the structure of umbilical arteries, with increases in wall areas and wall/lumen ratios.

Chronic cola drinking induces metabolic and cardiac alterations in rats.

AIM: To investigate the effects of chronic drinking of cola beverages on metabolic and echocardiographic parameters in rats. METHODS: Forty-eight male Wistar rats were divided in 3 groups and allowed to drink regular cola (C), diet cola (L), or tap water (W) ad libitum during 6 mo. After this period, 50% of the animals in each group were euthanized. The remaining rats drank tap water ad libitum for an additional 6 mo and were then sacrificed. Rat weight, food, and beverage consumption were measured regularly. Biochemical, echocardiographic and systolic blood pressure data were obtained at baseline, and at 6 mo (treatment) and 12 mo (washout). A complete histopathology study was performed after sacrifice. RESULTS: After 6 mo, C rats had increased body weight (+7%, P < 0.01), increased liquid consumption (+69%, P < 0.001), and decreased food intake (-31%, P < 0.001). C rats showed mild hyperglycemia and hypertriglyceridemia. Normoglycemia (+69%, P < 0.01) and sustained hypertriglyceridemia (+69%, P < 0.01) were observed in C after washout. Both cola beverages induced an increase in left ventricular diastolic diameter (C: +9%, L: +7%, P < 0.05 vs W) and volumes (diastolic C: +26%, L: +22%, P < 0.01 vs W; systolic C: +24%, L: +24%, P < 0.05 vs W) and reduction of relative posterior wall thickness (C: -8%, L: -10%, P < 0.05 vs W). Cardiac output tended to increase (C: +25%, P < 0.05 vs W; L: +17%, not significant vs W). Heart rate was not affected. Pathology findings were scarce, related to aging rather than treatment. CONCLUSION: This experimental model may prove useful to investigate the consequences of high consumption of soft drinks.

Subcutaneous connective tissue reaction to methacrylate resin-based and zinc oxide and eugenol sealers.

INTRODUCTION: An evaluation was made of the connective tissue reaction in rats after subcutaneous implantation of methacrylate resin-based sealers (EndoREZ [Ultradent Products, Inc, South Jordan, UT] with a polymerization accelerator and RealSeal [Sybron Dental Specialties, Orange, CA]) and Pulp Canal Sealer (Sybron Dental Specialties), a zinc oxide and eugenol-based sealer used as the control. METHODS: Silicone tubes containing the test materials were implanted in 24 Wistar rats. Solid silicone rods of the same size served as the negative controls. After 10, 30, and 90 days, the animals (n = 8 per period) were euthanized and the implants with surrounding tissues dissected and processed for routine histological evaluation. A four-category evaluation system was used to measure and record the microscopic observations according to the thickness of a fibrous capsule, the vascular changes, and the various types of inflammatory cells. RESULTS: Initially, a severe inflammatory reaction was observed of the soft tissues in direct contact with both EndoREZ/Accelerator and Real Seal. The severity decreased over time and was resolved at the end of the experiment. Pulp Canal Sealer showed a severe tissue reaction for all observation periods. The negative controls showed an initial mild to moderate inflammatory reaction. After 30 days, healthy fibrous connective tissue was observed, which increased over time. After 10 days, no statistically significant differences between the experimental groups were observed. After 90 days, EndoREZ and RealSeal were statistically significantly less toxic than Pulp Canal Sealer (p > 0.05). CONCLUSIONS: After 90 days, both methacrylate resin-based sealers were considered biologically acceptable when implanted in subcutaneous connective tissues of the rat. Pulp Canal Sealer remained toxic for the duration of the study.

Are kinking and coiling of carotid artery congenital or acquired?

Dolichoarteriopathies consist of tortuosity, kinking, or coiling of the extracranial carotid arteries. Some authors consider these alterations a consequence of atherosclerotic vessel remodeling, while others ascribe them to anatomical variations of embryological origin. The objective was to establish whether carotid dolichoarteriopathies belonged to a congenital origin or to acquired conditions. Color Doppler ultrasonography of neck vessels was performed in 885 participants, whose age ranged from 1-day-old infants to 90-year-old adults. Prevalence of kinking and coiling was evaluated, and it was related to the presence of cardiovascular risk factors. Prevalence of either kinking or coil of carotid arteries showed no increase with age, as it was comparable across all ages; furthermore, frequency of these alterations showed no relationship to cardiovascular risk factors nor to the presence of atheromatous plaques. These findings suggest that carotid dolichoarteriopathies are a result of alterations in embryological development rather than vascular remodeling secondary to aging and/or atherosclerosis.

Coronary intimal thickening in newborn babies and

We performed a morphological characterization of intimal thickenings in coronary arteries in the very early stages of life to obtain insights into initial coronary atherogenesis. We examined specimens from 67 infants who had died of noncardiac causes within their first year of life. Serially cut sections were stained with hematoxylin-eosin, Azan, Alcian blue, acetic orceine, and immunotypified for CD68, CD34, and alpha-smooth muscle (SM) actin. Substantial changes were detected in about 1 of 3 participants. Alterations ranged from focal areas with mild myointimal thickening to diffuse moderate thickening. In those lesions, smooth muscle cells (SMCs) showed loss of polarity, infiltrating the subendothelium, mostly with rupture of the internal elastic lamina and without neoangiogenesis. Morphometrically, in musculoelastic intimal thickenings, neointimal thickness averaged 58.3 +/- 17.8 microm, affecting 46% of the internal elastic membrane perimeter; lumen stenosis averaged 13.7% +/- 5.0%. These lesions can be present very early in life and SMCs seem to play an essential role.

Rat subcutaneous tissue response to modified Portland cement, a new mineral trioxide aggregate.

The purpose of this study was compare the biocompatibility of modified Portland cement (CPM) and mineral trioxide aggregate (MTA) in a subcutaneous rat model. Twenty-four male Wistar rats were used. Three silicon tubes were placed on the dorsal subcutaneous tissue of each animal: one tube contained MTA, one tube contained CPM and the other was an empty tube. The rats were sacrificed in 3 groups of 8 animals at 7, 14 and 30 postoperative days, respectively. Tissue samples were fixed in 10% buffered formalin, embedded in paraffin, and serial sections were cut and stained with hematoxylin and eosin, Masson Trichrome and Luna's stain. At day 7, the empty tubes displayed a mild inflammatory infiltrate. In the CPM group, an inflammatory infiltrate was observed with some eosinophils and immature connective tissue. The MTA group showed a similar infiltrate without eosinophils and presence of abundant necrotic tissue and numerous multinucleate foreign body giant cells. At day 14, the chronic infiltrate with eosinophils persisted when in contact with CPM. In the MTA group, necrosis and distant giant cells could still be seen. At day 30, all 3 groups showed mature fibrous collagenous tissue. These findings indicate a different response to the materials evaluated in this study. Although, MTA and CPM induced a chronic inflammatory infiltrate, necrosis and multinucleated foreign body giant cells predominated in the MTA group, while in the CPM group numerous eosinophils were seen at all the observational periods.

Carotid barochemoreceptor pathological findings regarding carotid plaque status and aging.

BACKGROUND: Carotid barochemoreceptor pathological lesions have been studied in animals, but few human necropsies have been performed. Therefore, data rely on case patients following surgery, radiotherapy and carotid endarterectomy. Almost no data are available regarding whether the effect of aging prevails over pathological conditions, despite the classic description that glomic fibrosis increases with age. OBJECTIVE: To morphometrically characterize the alterations of the carotid barochemoreceptors and their supplying arteries. METHODS: Patients (n=23) who had suffered and died from stroke, with and without complicated internal carotid atheromatosis, were divided by age (group 1: older than 80 years; group 2: 65 to 80 years; and group 3: younger than 65 years). Carotid segments were obtained at autopsy. The specimens were stained for light microscopy and immunohistochemistry. RESULTS: Carotid glomus presented from moderate-to-severe atrophy and fibrosis. A focal decrease in vascularization (CD34-positive) of the glomus (greater than 50%) was observed in areas of atrophy and fibrosis. Damaged nerve endings (S100 protein-positive) were observed at the media of the carotid sinus. Morphometric data showed no differences between groups for glomus area, number of type 1 and 2 cells, and the wall to lumen arteriole ratio. No statistical differences were demonstrated in the pathological findings of the carotid glomus when comparing complicated with noncomplicated plaques or age groups. CONCLUSION: Severe carotid chemoreceptor damage exists in patients who have died from stroke and suffered from carotid atheromatosis. These findings were independent from aging and plaque type. However, damage was correlated with a marked narrowing of the supplying arterioles as a consequence of hemodynamic and/or metabolic alterations (dyslipidemia, diabetes).

Perinatal and infant early atherosclerotic coronary lesions.

OBJECTIVE: Because the fetal origin of coronary artery lesions is controversial, early atherosclerotic coronary artery lesions in late fetal stillborns and infants, as well as the possible atherogenic role of maternal cigarette smoking, were studied. METHODS: Twenty-two fetal death and 36 sudden infant death syndrome victims were examined by autopsy. In 28 of 58 cases, the mothers were smokers. Serially cut sections of coronary arteries were stained for light microscopy and immunotypified for CD68, CD34, alpha-smooth muscle actin, proliferating cell nuclear antigen, c-fos and apoptosis. RESULTS: Multifocal coronary lesions were detected in 10 of 12 fetuses and in 15 of 16 infants whose mothers smoked. Arterial lesions in infants with nonsmoking mothers were observed in only five cases (two of 10 fetuses and three of 20 infants) (P<0.001). Alterations ranged from focal areas with mild myointimal thickening in prenatal life to early soft plaques in infants. Smooth muscle cells infiltrated into the subendothelium. These early lesions demonstrated c-fos gene activation in the smooth muscle cells of the media, and in some of these, positivity for apoptosis was observed, suggesting that c-fos overexpression may promote proliferation, as evidenced by proliferating cell nuclear antigen-positive cells. CONCLUSIONS: Early intimal alterations of the coronary arteries are detectable in the prenatal and infancy period, and may be significantly associated with maternal smoking.

Adriamycin-induced myocardial toxicity: new solutions for an old problem?

Adriamycin is a potent and broad-spectrum antineoplastic agent that plays a major role in cancer chemotherapy. Unfortunately, its use has been hampered by conventional toxicities and cardiotoxicity manifested by congestive cardiomyopathy. Adriamycin is particularly toxic to heart tissue and constitutes a major cause of morbidity and mortality due to its complex pathogenesis. In this review, the different forms of cardiotoxicity produced by adriamycin as well as the biochemical changes induced by this drug are summarized. Secondly, the current hypotheses proposed to explain adriamycin-induced myocardial damage (the iron and free-radical hypothesis, the metabolic hypothesis, the "unifying hypothesis" and apoptosis) and the attempts to reduce adriamycin-induced myocardial toxicity are discussed (e.g. dose limitation, close cardiac monitoring, alteration of dosage schedules, development of new anthracycline analogs, and the administration of protective agents and liposomal encapsulation). Finally, we summarized our own experimental and clinical experience in ameliorating and or preventing adriamycin-induced cardiotoxicity and the latest attempts to prevent and/or monitor cardiac function. According to this, a combination of usual doses of calcium antagonist drugs plus vitamins A and E seems advisable.

Unusual location in the left ventricular outflow tract and atypical symptoms of cardiac papillary fibroelastoma.

Cardiac papillary fibroelastoma, though potentially fatal, are rare benign tumours. Therefore, high index of suspicion is needed in order to identify these lesions and to limit complications at surgery or during long-term oral anticoagulation if the patient is not a surgical candidate. We present a case report of unusual location and presentation, highlighting the pathological findings. A 55-year-old white male, without risk factors for ischaemic cardiomyopathy, presented several episodes of chest pain. Nine years previously, the patient had had a convulsive attack without any organic neurological finding and was treated with different anticonvulsive drugs for five years. Echocardiography showed a mobile mass in the left ventricular outflow tract. The patient underwent surgical excision of the mass, which was later identified as cardiac papillary fibroelastoma.

Atherosclerotic plaque rupture and intraplaque hemorrhage do not correlate with symptoms in carotid artery stenosis.

OBJECTIVE: Previously we failed to demonstrate a correlation between plaque type and symptoms in 165 carotid endarterectomy specimens. The purpose of this study was to analyze the relation between the anatomy of the carotid plaques and the presence of symptoms in 281 carotid endarterectomy specimens. METHODS: The patients were 213 men (mean age, 68 years) and 68 women (mean age, 68.7 years), with symptomatic disease (n = 133) or asymptomatic disease (n = 148). Specimens were processed for histologic analysis and immunohistochemistry. RESULTS: Plaques were categorized as complicated or noncomplicated, and ruptured or nonruptured. Risk factors could not be correlated with any pathologic or immunohistochemical findings or between plaque type and clinical symptoms. CONCLUSIONS: Almost 70% of plaque specimens demonstrated thrombus, intraplaque hemorrhage, or both. Thrombosis was observed in one fourth of specimens, and intraplaque hemorrhage in almost two thirds of specimens. Sixty four percent of plaques demonstrated neovascularization. It was not possible to demonstrate that complicated plaques (plaque rupture, thrombosis, intraplaque hemorrhage) are associated with symptoms, and it appears that such plaques may occur at any time, irrespective of symptoms.