Renal arteriolar hyalinosis, not intimal thickening in large arteries, is associated with cardiovascular events in people with biopsy-proven diabetic nephropathy.

AIMS: Diabetic nephropathy, a pathologically diagnosed microvascular complication of diabetes, is a strong risk factor for cardiovascular events, which mainly involve arteries larger than those affected in diabetic nephropathy. However, the association between diabetic nephropathy pathological findings and cardiovascular events has not been well studied. We aimed to investigate whether the pathological findings in diabetic nephropathy are closely associated with cardiovascular event development. METHODS: This retrospective cohort study analysed 377 people with type 2 diabetes and biopsy-proven diabetic nephropathy, with a median follow-up of 5.9 years (interquartile range 2.0 to 13.5). We investigated how cardiovascular events were impacted by two vascular diabetic nephropathy lesions, namely arteriolar hyalinosis and arterial intimal thickening, and by glomerular and interstitial lesions. RESULTS: Of the 377 people with diabetic nephropathy, 331 (88%) and 295 (78%) had arteriolar hyalinosis and arterial intimal thickening, respectively. During the entire follow-up period, those with arteriolar hyalinosis had higher cardiovascular event rates in the crude Kaplan-Meier analysis than those without these lesions (P = 0.005, log-rank test). When fully adjusted for clinically relevant confounders, arteriolar hyalinosis independently predicted cardiovascular events [hazard ratio (HR) 1.99; 95% confidence interval (CI) 1.12, 3.86], but we did not find any relationship between arterial intimal thickening and cardiovascular events (HR 0.89; 95% CI 0.60, 1.37). Additionally, neither glomerular nor interstitial lesions were independently associated with cardiovascular events in the fully adjusted model. CONCLUSIONS: Arteriolar hyalinosis, but not intimal thickening of large arteries, was strongly associated with cardiovascular events in people with diabetic nephropathy.

Defining normal variables of right ventricular size and function in pulmonary hypertension: an echocardiographic study.

BACKGROUND: Right ventricular (RV) fractional area change and tricuspid annular plane systolic excursion (TAPSE) are recognised methods for assessing RV function. However, the way in which these variables are affected by varying degrees of pulmonary hypertension (PH) has not been well characterised. METHODS: RV end-systolic area (RVESA), RV end-diastolic area (RVEDA), pulmonary artery systolic pressure (PASP) and TAPSE were collected from a database of 190 patients who had been referred to the PH clinic for evaluation. RESULTS: The mean (SD) age of the study population was 56 (17) years; 82 men were included with a mean (SD) PASP of 54 (33) mm Hg (range 16-150), RVESA of 14 (9) cm(2), RVEDA of 24 (9) cm(2), RV fractional area change of 44 (18)% and TAPSE of 2.06 (0.69) cm. Receiver-operating characteristic curves identified TAPSE <2.01 cm, RV fractional area change <40.9%, RVESA >12.3 cm(2) and RVEDA >23.4 cm(2) as abnormal values with PH. Finally stratification of patients into sub-groups according to their PASP allowed means and standard deviations to be reported for each echocardiographic variable. CONCLUSION: This analysis provides a range of normal variables of RV size and function, not previously published, that can be used in routine evaluation and follow-up of patients with PH.

Pharmacological brain cooling with indomethacin in acute hemorrhagic stroke: antiinflammatory cytokines and antioxidative effects.

We evaluated the effects of a novel pharmacological brain cooling (PBC) method with indomethacin (IND), a nonselective cyclooxygenase inhibitor, without the use of cooling blankets in patients with hemorrhagic stroke. Forty-six patients with hemorrhagic stroke (subarachnoid hemorrhage; n = 35, intracerebral hemorrhage; n = 11) were enrolled in this study. Brain temperature was measured directly with a temperature sensor. Patients were cooled by administering transrectal IND (100 mg) and a modified nasopharyngeal cooling method (positive selective brain cooling) initially. Brain temperature was controlled with IND 6 mg/kg/day for 14 days. Cerebrospinal fluid concentrations of interleukin-1beta (CSF IL-1beta) and serum bilirubin levels were measured at 1, 2, 4, and 7 days. The incidence of complicating symptomatic vasospasm after subarachnoid hemorrhage was lower than in non-PBC patients. CSF IL-1beta and serum bilirubin levels were suppressed in treated patients. IND has several beneficial effects on damaged brain tissues (anticytokine, free radical scavenger, antiprostaglandin effects, etc.) and prevents initial and secondary brain damage. PBC treatment for hemorrhagic stroke in patients appears to yield favorable results by acting as an antiinflammatory cytokine and reducing oxidative stress.

Controlled normothermia during ischemia is important for the induction of neuronal cell death after global ischemia in mouse.

A stable model of neuronal damage after ischemia is needed in mice to enable progression of transgenic strategies. We performed transient global ischemia induced by common carotid artery occlusions with and without maintaining normal rectal temperature (Trec) in order to determine the importance of body temperature control during ischemia. We measured brain temperature (Tb) during ischemia/reperfusion. Mice with normothermia (Trec within +/- 1 degrees C) had increased mortality and neuronal cell death in the CA1 region of hippocampus, which did not occur in hypothermic animals. If the Trec was kept within +/- 1 degrees C, the Tb decreased during ischemia. After reperfusion, Tb in the normothermia group developed hyperthermia, which reached > 40 degrees C and was > 2 degrees C higher than Trec. We suggest that tightly controlled normothermia and prevention of hypothermia (Trec) during ischemia are important factors in the development of a stable neuronal damage model in mice.

Positive selective brain cooling method: a novel, simple, and selective nasopharyngeal brain cooling method.

Brain damage is worsened by hyperthermia and prevented by hypothermia. Conventional hypothermia is a non-selective brain cooling method that employs cooling blankets to achieve surface cooling. This complicated method sometimes induces unfavorable systemic complications. We have developed a positive selective brain cooling (PSBC) method to control brain temperature quickly and safely following brain injury. Brain temperature was measured in patients with a ventriculostomy CAMINO catheter. A Foley balloon catheter was inserted to direct chilled air (8 to 12 L/min) into each side of the nasal cavity. The chilled air was exhaled through the oral cavity. In most patients, PSBC maintained normal brain temperature. This new technique provides quick induction of brain temperature control and does not require special facilities.

Inducible virus-mediated expression of a foreign protein in suspension-cultured plant cells.

Although suspension-cultured plant cells have many potential merits as sources of useful proteins, the lack of an efficient expression system has prevented using this approach. In this study, we established an inducible tomato mosaic virus (ToMV) infection system in tobacco BY-2 suspension-cultured cells to inducibly and efficiently produce a foreign protein. In this system, a modified ToMV encoding a foreign protein as replacement of the coat protein is expressed from stably transformed cDNA under the control of an estrogen-inducible promoter in transgenic BY-2 cells. Estrogen added to the culture activates an estrogen-inducible transactivator expressed constitutively from the transgene and induces transcription and replication of viral RNA. In our experiments, accumulation of viral RNA and expression of green fluorescent protein (GFP) encoded in the virus were observed within 24 h after induction. The amount of GFP reached approximately 10% of total soluble protein 4 d after induction. In contrast, neither viral RNA nor GFP were detected in uninduced cells. The inducible virus infection system established here should be utilized not only for the expression of foreign proteins, but also for investigations into the viral replication process in cultured plant cells.

CSF hypocretin-1/orexin-A concentrations in patients with subarachnoid hemorrhage (SAH).

The aim of this study was to examine the role of the hypothalamic hypocretin/orexin system in complications of delayed ischemic neuronal deficit (DIND) resulting from symptomatic vasospasm in patients with aneurysmal subarachnoid hemorrhage (SAH). CSF hypocretin-1/orexin-A levels were measured in 15 SAH patients. DIND complications occurred in seven patients with symptomatic vasospasm. Hypocretin-1/orexin-A levels were low in SAH patients during the 10 days following the SAH event. CSF hypocretin-1/orexin-A levels were lower in patients with DIND complications than in those who did not develop DIND. A significant transient decline in CSF hypocretin-1/orexin-A levels was also observed at the onset of DIND in all patients with symptomatic vasospasm. The reduced hypocretin/orexin production observed in SAH patients may reflect reduced brain function due to the decrease in cerebral blood flow. These results, taken together with recent experimental findings in rats that indicate hypocretin receptor 1 (orexin 1 receptor) mRNA and protein are elevated following middle cerebral artery occlusion, suggest that a reduction in hypocretin/orexin production in SAH and DIND patients is associated with alterations in brain hypocretin/orexin signaling in response to ischemia.

Strength, workload, anaerobic intensity and the immune response to resistance exercise in women.

AIM: The mechanism linking exercise intensity to the magnitude of the immune response is not completely understood. The purpose of this investigation was to determine whether the immune response to resistance exercise was associated with (1) changes in workload or (2) anaerobic exercise intensity. METHODS: Previously untrained women underwent 6 months of resistance training for lower and upper body (TOTAL, n = 34) or for upper body alone (UPPER, n = 30). Lymphocyte subsets [T (CD3+), CD4+, CD8+, NK and B], functional markers (CD45RA+ and CD45RO+), and mitogen (phytohemagglutinin-M, concanavalin A and pokeweed mitogen) and superantigen (staphylococcus a. cowans)-stimulated proliferation were measured from blood samples collected pre- and post-exercise for a squat resistance exercise consisting of six sets of 10 repetitions at 75% of one repetition maximum. This protocol was performed before (T0) and after 3 (T3) and 6 months (T6) of training. RESULTS: Lymphocyte recruitment to the circulation and proliferation following resistance exercise did not differ between training groups at any time, although the TOTAL group performed at a higher workload as training progressed. With respect to anaerobic intensity, exercise-induced increases in NK, CD4+, CD8+ and B lymphocyte concentrations were 42 (P = 0.07), 76 (P < 0.05), 72 (P < 0.05) and 242% (P < 0.01) greater in women in the highest compared with the lowest post-exercise lactate quartiles. Lymphocyte proliferation did not differ between lactate quartiles. CONCLUSIONS: Anaerobic intensity, rather than increased strength and workload, is associated with the number of lymphocytes recruited to the circulation, but not T and B cell proliferation responses.

Expression of tumor necrosis factor alpha (TNFalpha) following transient cerebral ischemia.

It has been considered that tumor necrosis factor alpha (TNFalpha) is participated in the Alzheimer's, and Parkinson's diseases, brain injury and brain ischemia. However, expression of TNFalpha after brain ischemia has not been demonstrated in detail. Therefore we examined the cellular expression of TNFalpha during and after transient middle cerebral artery occlusion (tMCAO) in mice by use of reverse transcriptase-polymerase chain reaction and immunohistochemical technique. TNFalpha mRNA expression was gradually increased in the neocortex of the ipsilateral hemisphere during ischemia and peaked at 1 hour after reperfusion. Then, the mRNA expression decreased and peaked again at 24 hours after reperfusion. TNFalpha-like immunoreactivities were observed in the process such as dendrite of neuron slightly before ischemia, and markedly increased in neurons in addition to the process of the ipsilateral hemisphere at 1 and 24 hours after ischemia. The results suggest that the expression of TNFalpha is up-regulated in the neurons after tMCAO. TNFalpha may induce ischemic neuronal cell death during ischemic insult.

Peroxynitrite and caspase-3 expression after ischemia/reperfusion in mouse cardiac arrest model.

NO is a putative neurotransmitter and neuromodulator in the brain. NO is not functioning as a direct neurotoxin. NO with the superoxide radical product peroxynitrite (ONOO-) is much more cytotoxic under tissue impairment conditions. Caspase-3, a potent effector of apoptosis that is triggered via several different signaling pathways, may play a very important role in neuronal cell death caused by various brain injuries. The relationship between mouse caspase-3 and peroxynitrite remains unclear. In the present study, we examined the in vivo expression of 3-nitrotyrosine (a metabolite of peroxinitrite) and caspase-3 after cerebral ischemia produced in a global ischemia model using mice (i.e., a cardiac arrest model). 3-nitrotyrosine immunoreactivity was detected in neuronal cells in the hippocampal dentate nucleus, and cortical regions starting at 12 hrs after ischemia. In particular, numerous neuronal cells were highly immunoreactive for 3-nitrotyrosine in the cortical regions. In hippocampal CA1 pyramidal neurons, 3-nitrotyrosine immunoreactivity was detected from 24 hrs. Caspase-3 immunopositive cells were observed in approximately the same area in which the positive reaction to the anti-nitrotyrosine antibody was observed. These results provide direct evidence for the induction of 3-nitrotyrosine and caspase-3 expression in vivo in an ischemia model using mice. The present findings suggest that peroxynitrite generated by cerebral ischemia/ reperfusion was strongly cytotoxic and induced neuronal cell death (apoptosis) mediated by caspase-3.

Evaluation of neuronal cell death after a new global ischemia model in infant mice.

For the first time we set up a new model for global ischemia in the infant mice, and time-dependent changes of the blood-brain barrier (BBB) disruption and neuronal cell death were investigated in detail. Infant C57/B16 mice (postnatal 13 days) were anesthetized with inhalation of sevoflurane in N2O/O2 (70/30%) and were subjected to global ischemia by bilateral common carotid artery occlusion (CCAO) for 25 minutes. Disruption of BBB was noted at 4 hours and increased up to 24 hours after the injection of 2% Evan's Blue in the transient CCAO (tCCAO) model. Evaluation of neuronal cell death was determined with toluidine blue staining. Morphological changes of neurons after tCCAO were clearly observed in the hippocampal CA1 region but were slightly detected in the CA3 region. However, there were no morphological changes in the hippocampal dentate gyrus, the neocortex, the striatum and the hypothalamus. The number of survival neurons in the CA1 was significantly decreased at 2 days and sustained up to 4 days after tCCAO. These data indicate that this method is very useful to induce selective vulnerability in mouse hippocampus, and it provides a reliable ischemic model in infant mice.

Hippocampal heme oxigenase-1 in a murine cardiac arrest model.

Heme oxygenase-1 (HO-1) is a member of the heat shock protein family (HSP-32). It responds to thermal stress in cultured glial cells. To our knowledge. nothing is known about the expression and response of the HO-1 in cerebral ischemia. Therefore, we show here the induction of HO-1 in the brain of mice after global cerebral ischemia. HO-1-like immunoreactivity was detected at 12, 24, and 48 hours after ischemia recirculation. The HO-1-like immunoreactive cells were observed in astrocytes in the hippocampal dentate gyrus and CA1. The peak level of HO-1-like immunoreactivity was found 48 hours after the recirculation. HO-1-like immunoreactivity was observed in GFAP-positive astrocytes by use of a double immunostaining method. These results provide direct evidence for the induction and localization of HO-1 immunoreactivity in vivo in a mouse cerebral ischemia. We suggest that HO-1, produced in astrocytes after ischemia-recirculation, may directly affect neurons to protect from cell death.

Neuroprotective effect from ischemia and direct free radical scavenging activity of Choto-san (kampo medicine).

Choto-san is a kampo medicine that is widely used in patients with cerebral infarction, but the details of its mechanism of action remain unclear. We examined the neuroprotective effects of Choto-san using an experimental cerebral ischemia model (i.e., a rat cardiac arrest model). We also investigated the ability of Choto-san to eliminate or inhibit the activity of free radicals. It was found that Choto-san significantly prevents delayed neuronal cell death after ischemic reperfusion. Electron spin resonance demonstrated that the formation of hydroxyl- and superoxide-DMPO spin adducts were inhibited by Choto-san. The results of this study indicated that Choto-san prevents delayed neuronal cell death in the hippocampal CA1 region after ischemia. Direct free radical scavenging activity is among the pharmacological effects of Choto-san.

Suppression of oxidative stress after transient focal ischemia in interleukin-1 knock out mice.

Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clarify this central issue, mice that were gene deficient both IL-1alpha and beta (IL-1 KO) and wild-type mice were subjected to 1 hour transient middle cerebral artery occlusion (tMCAO). The concentration of 8-hydroxy deoxyguanosine (8OHdG) which is considered to be a reliable oxidative DNA damage by superoxide anion, in brain and of total nitric oxide (NO) in plasma were determined by use of HPLC. Twenty-four hours after tMCAO, the ratio of 8OHdG to dG in the ipsilateral hemisphere of wild-type mice were 2.24 x 10(-3) and 4.41 x 10(-3) in the neocortex and striatum, respectively. The concentration of 8OHdG in the ipsilateral hemisphere of the wild-type mice was higher than that of the IL-1 KO mice. The concentration of total NO in the plasma of IL-1 KO mice was also lower than that of the wild-type 24 hours after tMCAO. These results strongly suggest that IL-1 is participated in generating reactive oxygen spices and it aggravates and induces the ischemic neuronal cell death.(183 words).

Transient elevation of serum bilirubin (a heme oxygenase-1 metabolite) level in hemorrhagic stroke: bilirubin is a marker of oxidant stress.

Bilirubin (Bil) is the end product of heme catabolism. The production of Bil reflects heme oxygenase-1 expression in response to oxidative stress in various diseases. To assess the role of Bil as a marker of oxidative stress in cases of brain damage, we measured serum Bil concentrations in patients with hemorrhagic stroke. Serum levels of total Bil were measured in 20 subarachnoid hemorrhage patients with symptomatic vasospasms and in 23 patients with intracerebral hemorrhage; concentrations were measured every day for 14 consecutive days. Serum Bil levels were significantly elevated in the early phases in both groups. Moreover, transient elevation was observed on the day prior to the observation of clinical manifestations of symptomatic vasospasm after SAH. Bil, known to be a powerful antioxidant, was induced after hemorrhagic stroke, reflecting the intensity of oxidative stress. Plasma Bil concentrations might serve as a useful marker of oxidative stress in hemorrhagic stroke patients.

Circadian rhythm of blood pressure is transformed from a dipper to a non-dipper pattern in shift workers with hypertension.

Shift workers make great use of health care services because they are associated with increased cardiovascular morbidity and mortality. Whether the circadian rhythm of blood pressure rapidly adapts to shift work is controversial. It is unknown if shift work has adverse effects on blood pressure in patients with hypertension. To evaluate the effects of shift work, we examined 12 male shift workers with untreated hypertension aged 53.6 +/- 2.5 years. Twenty-four hour ambulatory blood pressure monitoring was performed three times as follows: the last day of a 4-day period of day shifts (09.00 to 21.00), the first day of a 4-day period of night shifts (21.00 to 09.00), and the fourth day of night shifts (21.00 to 09.00). Blood pressure at night-time dropped significantly in the day-shift workers, showing a dipper pattern. Average differences in blood pressure in the sleep-wake cycle were decreased by 8.5% at the beginning of night shift work showing a non-dipper pattern. After 4 days the pattern was completely reversed to a dipper pattern. The results indicate that the circadian blood pressure pattern is changed from a dipper to a non-dipper pattern on the first day of the night shift and reverses to a dipper pattern within a few days. We suggest that night shift work may have unfavourable effects on blood pressure in patients with hypertension.

Vascular endothelial growth factor mRNA synthesis by peripheral blood mononuclear cells in patients with acute myocardial infarction.

We studied vascular endothelial growth factor (VEGF) mRNA synthesis by peripheral blood mononuclear cells (PBMCs) in 30 patients with acute myocardial infarction (AMI) and 20 healthy individuals. PBMCs were isolated from all patients on days 3 and 14 after the onset of aMI, and from all of control individuals. To prepare samples containing identical amounts of GAPDH cDNA, competitive PCR was performed by co-amplifying serial dilutions of GAPDH mutant templates. Next, to measure VEGF cDNA quantitatively in the samples containing identical amounts of GAPDH, we also used competitive PCR by co-amplifying mutant templates of VEGF. The serum VEGF concentrations on day 14 in patients with aMI were measured by an ELISA method. Higher levels of VEGF mRNA in PBMCs were present on day 14 than either on day 3 or in the control group. Serum VEGF concentrations correlated with the VEGF mRNA levels of PBMCs on day 14. Peak serum CK levels correlated well with VEGF mRNA levels of PBMCs on day 14. The present findings suggest that PBMCs may be one of the candidates responsible for elevated circulatory VEGF protein following aMI. In addition, VEGF mRNA may be overexpressed in PBMCs in response to cardiac muscle damage.

Effects of therapeutic doses of human atrial natriuretic peptide on load and myocardial performance in patients with congestive heart failure.

The benefits of atrial natriuretic peptide (ANP) in patients with congestive heart failure (CHF) have been demonstrated. However, the myocardial actions of ANP remain unclear. Using relatively load-insensitive left ventricular pressure-volume analysis, the myocardial and load-altering actions of ANP in patients with moderate CHF were studied. After obtaining steady-state data using micromanometers and conductance catheters, ANP was infused in 9 patients with CHF at 0.01 and 0.1 microg/kg/min for 30 minutes, respectively. Hemodynamic variables, plasma ANP, and cyclic guanosine monophosphate (cGMP) levels were determined before and 30 minutes after each ANP infusion. ANP at 0.01 microg/kg/min increased plasma ANP and cGMP levels from 73 +/- 34 to 139 +/- 34 pg/ml and from 4 +/- 1 to 8 +/- 2 pmol/ml, respectively. ANP infusion caused a significant decrease in end-systolic pressure without any changes in heart rate. End-diastolic pressure was significantly decreased but there was no significant change in left ventricular end-diastolic volume. The time constant for isovolumetric relaxation was decreased. ANP infusion at 0.1microg/kg/min caused further decreases in end-systolic pressure, end-diastolic pressure and volume, and the time constant for isovolumetric relaxation (p <0.05) without any changes in heart rate. The slope of the end-systolic pressure-volume relation was increased from 1.3 +/- 0.2 to 1.6 +/- 0.3 mm Hg/ml (p <0.05), indicating increased contractility. Plasma ANP and cGMP levels were increased to 422 +/- 44 pg/ml and 16 +/- 3 pmol/ml, respectively. Thus, ANP infusion increased cGMP generation, decreased afterload and preload, and improved left ventricular systolic and diastolic function.