Hideaki Higashino

Study on Application of Static Magnetic Field for Adjuvant Arthritis Rats

In order to examine the effectiveness of the application of static magnetic field (SMF) on pain relief, we performed a study on rats with adjuvant arthritis (AA). Sixty female Sprague–Dawley (SD) rats (age: 6 weeks, body weight: approximately 160 g) were divided into three groups [SMF-treated AA rats (Group I), non-SMF-treated AA rats (Group II) and control rats (Group III)]. The SD rats were injected in the left hind leg with 0.6 mg/0.05 ml Mycobacterium butyrium to induce AA. The rats were bred for 6 months as chronic pain model. Thereafter, the AA rats were or were not exposed to SMF for 12 weeks. We assessed the changes in the tail surface temperature, locomotor activity, serum inflammatory marker and bone mineral density (BMD) using thermography, a metabolism measuring system and the dual-energy X-ray absorptiometry (DEXA) method, respectively. The tail surface temperature, locomotor activity and femoral BMD of the SMF-exposed AA rats were significantly higher than those of the non-SMF-exposed AA rats, and the serum inflammatory marker was significantly lower. These findings suggest that the pain relief effects are primarily due to the increased blood circulation caused by the rise in the tail surface temperature. Moreover, the pain relief effects increased with activity and BMD of the AA rats.

PARADOXICALLY ENHANCED HEART TOLERANCE TO ISCHAEMIA IN TYPE 1 DIABETES AND ROLE OF INCREASED OSMOLARITY

1 There is considerable controversy regarding the tolerance of diabetic hearts to ischaemia and the underlying mechanisms responsible for the increased heart tolerance to ischamia remain uncertain. In the present study, we observed, in vitro, type 1 diabetic heart responses to ischaemia and reperfusion at different degrees of hyperglycaemia. In addition, the possible role of increased osmolarity in cardioprotection due to hyperglycaemia was evaluated. 2 Hearts from 3 week streptozocin‐induced diabetic rats were isolated and perfused in a Langendorff apparatus and subjected to 30 min ischaemia and 30 min reperfusion. Cardiac function and the electrocardiogram were recorded. Myocardial content of osmolarity associated heat shock protein (hsp) 90, heme oxygenase (HO)‐1 and anti‐oxidant enzymes were determined in diabetic or hyperosmotic solution‐perfused hearts using western blot. The hsp90 inhibitor 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG; 2 × 10−7 mol/L) or the nitric oxide synthase (NOS) inhibitor Nω‐nitro‐l‐arginine methyl ester (1 × 10−5 mol/L) was added to the perfusate to observe the effects of hsp90 inhibition and hsp90‐associated endothelial NOS on ischaemic responses of diabetic hearts. 3 Compared with normal control rats, diabetic hearts with severe hyperglycaemia (blood glucose > 20 mmol/L) showed markedly improved postischaemic heart function with fewer reperfusion arrhythmias. Mild hyperglycaemia (< 12 mmol/L) exhibited no significant cardioprotection. 4 Elevated expression of hsp90 accompanied the enhanced resistance to ischaemia in diabetic hearts, which was abrogated by 17‐AAG. In the presence of the NOS inhibitor, heart function was preserved, whereas reperfusion arrhythmias were increased in diabetes. Diabetic hearts also had markedly elevated HO‐1 and catalase, with no significant change in superoxide dismutase. Hyperosmotic perfusion with glucose or mannitol also increased myocardial hsp90 and catalase. 5 The present findings reveal that heart resistance to ischaemia is increased in short‐term type 1 diabetes with severe hyperglycaemia. Elevated osmolarity caused by significant hyperglycaemia may contribute to the enhanced myocardial activity against oxidative injury during ischaemia and reperfusion.

SERUM NITRIC OXIDE METABOLITE (NOX) LEVELS IN HYPERTENSIVE PATIENTS AT REST: A COMPARISON OF AGE, GENDER, BLOOD PRESSURE AND COMPLICATIONS USING NORMOTENSIVE CONTROLS

1 Hypertensive patients have pathophysiological changes such as atherosclerosis, endothelial dysfunction and inflammations. The patients’ serum nitric oxide metabolite (nitrate/nitrite; NOx) levels were measured in peripheral blood using normotensive controls for comparison. 2 The NOx levels in 175 hypertensive patients with or without comorbid diseases (aged 37–95 years; average 50.6 ± 0.8 years) were compared with those in 80 normotensive controls (aged 25–73 years; average 37.1 ± 1.8 years). 3 The NOx levels increased with age in both the normotensive and hypertensive women, but not in men. No difference was noted in the NOx levels between the normotensive and hypertensive patients without comorbid diseases. The mean value of NOx in male hypertensive patients aged under 50 years was close to that of female patients aged 51–60 years. Hypertensive males aged 61–70 years showed almost the same NOx levels as those of female patients aged over 81 years. A male group of hypertensive patients with diabetes, hyperlipaemia and renal disorder had a significantly higher NOx level compared with a normotensive control group. However, in female groups, only hypertensive patients with hyperlipaemia showed higher serum NOx values compared with the normotensive group. 4 These findings suggest that: (i) the occurrence of NOx in the serum is not solely the outcome of high blood pressure; (ii) higher serum NOx levels in older women are because of an oestrogen deficiency‐induced cardiovascular disease; (iii) ageing effects on the circulation system are more apparent in men than in women; and (iv) measurement of NOx levels in the serum is helpful for understanding the pathological progress in male hypertensive patients with diseases such as diabetes mellitus, hyperlipaemia and renal disorder.

Acetylsalicylic acid provides cerebrovascular protection from oxidant damage in salt-loaded stroke-prone rats

Inflammatory processes may play a pivotal role in the pathogenesis of cerebrovascular injury in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP). Recent reports revealed that acetylsalicylic acid (aspirin) has anti-oxidative properties and elicits nitric oxide release by a direct activation of the endothelial NO synthase. The present study was designed to determine whether low-dose aspirin might prevent cerebrovascular injury in salt-loaded SHRSP by protecting oxidative damage. Nine-week-old SHRSP were fed a 0.4% NaCl or a 4% NaCl diet with or without treatment by naproxen (20 mg/kg/day), salicylic acid (5 mg/kg/day), or aspirin (5 mg/kg/day) for 5 weeks. Blood pressure, blood brain barrier impairment, mortality, and the parameters of cerebrovascular inflammation and damage were compared among them. High salt intake in SHRSP significantly increased blood brain barrier impairment and early mortality, which were suppressed by treatment with aspirin independent of changes in blood pressure. Salt loading significantly increased superoxide production in basilar arteries of SHRSP, which were significantly suppressed by treatment with aspirin. Salt loading also significantly decreased NOS activity in the basilar arteries of SHRSP, which were significantly improved by treatment with aspirin. At 5 weeks after salt loading, macrophage accumulation and matrix metalloproteinase-9 activity at the stroke-negative area in cerebral cortex of SHRSP were significantly reduced by treatment with aspirin. These results suggest that low-dose aspirin may exert protective effects against cerebrovascular inflammation and damage by salt loading through down-regulation of superoxide production and induction of nitric oxide synthesis.

In Vivo Diagnostic Imaging Using Micro-CT: Sequential and Comparative Evaluation of Rodent Models for Hepatic/Brain Ischemia and Stroke

Background There is an increasing need for animal disease models for pathophysiological research and efficient drug screening. However, one of the technical barriers to the effective use of the models is the difficulty of non-invasive and sequential monitoring of the same animals. Micro-CT is a powerful tool for serial diagnostic imaging of animal models. However, soft tissue contrast resolution, particularly in the brain, is insufficient for detailed analysis, unlike the current applications of CT in the clinical arena. We address the soft tissue contrast resolution issue in this report. Methodology We performed contrast-enhanced CT (CECT) on mouse models of experimental cerebral infarction and hepatic ischemia. Pathological changes in each lesion were quantified for two weeks by measuring the lesion volume or the ratio of high attenuation area (%HAA), indicative of increased vascular permeability. We also compared brain images of stroke rats and ischemic mice acquired with micro-CT to those acquired with 11.7-T micro-MRI. Histopathological analysis was performed to confirm the diagnosis by CECT. Principal Findings In the models of cerebral infarction, vascular permeability was increased from three days through one week after surgical initiation, which was also confirmed by Evans blue dye leakage. Measurement of volume and %HAA of the liver lesions demonstrated differences in the recovery process between mice with distinct genetic backgrounds. Comparison of CT and MR images acquired from the same stroke rats or ischemic mice indicated that accuracy of volumetric measurement, as well as spatial and contrast resolutions of CT images, was comparable to that obtained with MRI. The imaging results were also consistent with the histological data. Conclusions This study demonstrates that the CECT scanning method is useful in rodents for both quantitative and qualitative evaluations of pathologic lesions in tissues/organs including the brain, and is also suitable for longitudinal observation of the same animals.

Involvement of thromboxane A2 receptor in the cerebrovascular damage of salt-loaded, stroke-prone rats

Background Inflammatory processes may play a pivotal role in the pathogenesis of cerebrovascular injury in salt-loaded, stroke-prone, spontaneously hypertensive rats (SHRSP). Thromboxane A2 (TP) receptor stimulation by 8-iso-prostaglandin F2α (8-iso-PGF2α) is involved in the process of vascular inflammation. Objective In the present study, we examined the involvement of TP receptor in the development of cerebrovascular damage in salt-loaded SHRSP. Methods Nine-week-old SHRSP were fed a 0.4% NaCl or a 4% NaCl diet with or without ONO-8809 treatment (a TP receptor antagonist) for 5 weeks. Blood pressure, mortality, and the parameters of cerebrovascular inflammation and damage were compared between the groups. Moreover, we examined the effect of 8-iso-PGF2α infusion on cerebrovascular injury of SHRSP. Results High salt intake in SHRSP significantly increased blood–brain barrier impairment and early mortality, which were suppressed by ONO-8809 treatment independent of changes in blood pressure. Salt loading also significantly increased superoxide production in basilar arteries of SHRSP, which was suppressed by ONO-8809 treatment. Macrophage accumulation and matrix metalloproteinase-9 (MMP-9) activity in the stroke-negative area in the contralateral cerebral cortex to the stroke lesion of salt-loaded SHRSP and 8-iso-PGF2α–treated SHRSP were significantly reduced by ONO-8809 treatment. The ONO-8809 treatment prevented thinning of the vessel layer in cerebral arterioles of salt-loaded SHRSP and 8-iso-PGF2α–treated SHRSP. Conclusions These results suggest that TP receptor stimulation by 8-iso-PGF2α may involve salt loading-induced stroke through activation of cerebrovascular inflammation and damage.

Impaired heart function and noradrenaline release after ischaemia in stroke-prone spontaneously hypertensive rats.

1. Stroke‐prone spontaneously hypertensive rats (SHRSP) are a strain of rat that exhibit severely high blood pressure and stroke attacks at an early age, but their heart function in vitro has seldom been studied in detail. Although the activity of the sympathetic nervous system is known to increase after myocardial ischaemia, there is little information about the cardiac release of noradrenaline (NA) associated with heart function after ischaemia in SHRSP. The aim of the present study was to examine heart function and cardiac NA release after ischaemia in SHRSP.

Paradoxical effects of streptozotocin‐induced diabetes on endothelial dysfunction in stroke‐prone spontaneously hypertensive rats

Non‐Technical Summary  Elevated blood glucose is generally regarded as one of the risk factors that lead to coronary heart disease in patients with type 2 diabetes. However, our studies show that after inducing short‐term damage, high blood glucose subsequently provides paradoxical protection for vessel function of animals with high blood pressure. Vessels can adapt to sustained high blood glucose and produce different stress proteins to counteract, to some extent, the damage brought about by hypertension. The results help us understand part of the basis for vessel adaptation in diabetes. The implication for treatment of diabetes is that if the patients have long‐standing diabetes and established cardiovascular disease, the target of blood glucose lowering should be less stringent and reached gradually to avoid abrupt cancellation of the pre‐existing adaptations.

Preserved postischemic heart function in sucrose-fed type 2 diabetic OLETF rats

Cardiovascular disease is one of the most important causes of morbidity and mortality in diabetes mellitus, but there has been controversy over functional impairment of diabetic hearts and their tolerance to ischemia. We studied ischemic heart function in type 2 diabetic rats with different degrees of hyperglycemia and its relationship with cardiac norepinephrine release. Otsuka Long-Evans Tokushima Fatty rats (OLETF) and age-matched Long-Evans Tokushima Otsuka normal rats (LETO) were used. One group of OLETF rats was given 30% sucrose in drinking water (OLETF-S). Hearts were isolated and perfused in a working heart preparation and subjected to 30 min ischemia followed by 40 min reperfusion at age of 12 months. Hemodynamics and coronary norepinephrine overflow were examined. Fasting plasma glucose in OLETF increased markedly at 12 months and sucrose administration exacerbated hyperglycemia in diabetic rats (LETO 6.6 +/- 0.5, OLETF 8.3 +/- 0.7, OLETF-S 15.0 +/- 1.7 mmol/L, P < 0.01). Basic cardiac output in OLETF was decreased as compared with LETO and OLETF-S (LETO 29.4 +/- 2.5, OLETF 24.0 +/- 2.4, OLETF-S 27.0 +/- 0.9 ml/min/g, P < 0.05) and remained very low after ischemia, while in OLETF-S it was well preserved (OLETF 4.2 +/- 2.1, OLETF-S 13.7 +/- 2.6 ml/min/g, P < 0.01). Correspondently, cardiac norepinephrine released during ischemia and reperfusion was lower in OLETF-S (OLETF 2.3 +/- 1.0, OLETF-S 0.7 +/- 0.1 pmol/ml, P < 0.01). Thus, OLETF hearts were more vulnerable to ischemia but sucrose feeding rendered their hearts resistant to ischemia. Less norepinephrine release may play a role in preventing postischemic functional deterioration in sucrose-fed diabetic hearts.

Chymase inhibition improves vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats

Objective: To clarify the role of chymase in hypertension, we evaluated the effect of a chymase inhibitor, TY-51469, on vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats (SHR-SP). Methods: SHR-SP were treated with TY-51469 (1 mg/kg per day) or placebo from 4 to 12 weeks old or until death. Wistar–Kyoto rats were used as a normal group. Results: SBP was significantly higher in both the placebo and TY-51469 groups than in the normal group, but there was no significant difference between the two treatment groups. Plasma renin, angiotensin-converting enzyme activity and angiotensin II levels were not different between the placebo and TY-51469 groups. In contrast, vascular chymase-like activity was significantly higher in the placebo than in the normal group, but it was reduced by TY-51469. Acetylcholine-induced vascular relaxation was significantly higher in the TY-51469 group than in the placebo group. There was significant augmentation of the number of monocytes/macrophages and matrix metalloproteinase-9 activity in aortic tissue from the placebo group compared with the normal group, and these changes were attenuated by TY-51469. There were also significant increases in mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-&agr; in the placebo group that were attenuated by TY-51469. Cumulative survival was significantly prolonged in the TY-51469 group compared with the placebo group. Conclusion: Chymase might play an important role in vascular dysfunction via augmentation both of matrix metalloproteinase-9 activity and monocyte/macrophage accumulation in SHR-SP, and its inhibition may be useful for preventing vascular remodeling and prolonging survival.