Masaki Tabuchi

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.

Ecklonia cava polyphenol protects the liver against ethanol-induced injury in rats.

BACKGROUND The development of alcoholic liver disease is a complex process that involves both the parenchymal and non-parenchymal cells of the liver. We examined the effect of an Ecklonia cava extract on ethanol-induced liver injury. METHODS Isolated hepatocytes and hepatic stellate cells (HSCs) were incubated with ethanol. Ecklonia cava polyphenol (ECP) was added to the cultures that had been incubated with ethanol. Male Wistar rats were fed a diet that included 0.02% or 0.2% ECP or no ECP. For a period of 3 weeks, the animals were given drinking water containing 5% ethanol and were also treated with carbon tetrachloride (CCl4) (0.1 ml/kg of body weight). RESULTS In the cultured hepatocytes, the ECP treatment suppressed the ethanol-induced increase in cell death by maintaining intracellular glutathione (GSH) levels. In HSCs, ECP treatment suppressed the ethanol-induced increases in type I collagen and α-smooth muscle actin expression by maintaining intracellular levels of reactive oxygen species and GSH. We examined the effects of ECP on serum AST and ALT activity, as well as the progression of liver fibrosis in rats treated with ethanol and CCl4. ECP treatment suppressed plasma AST and ALT activities in the ethanol- and CCl4-treated rats. ECP treatment fully protected the rats against ethanol- and CCl4-induced liver injury. GENERAL SIGNIFICANCE ECP may be a candidate for preventing ethanol-induced liver injury.

Interleukin-1β Accelerates the Onset of Stroke in Stroke-Prone Spontaneously Hypertensive Rats

High blood levels of inflammatory biomarkers and immune cells in stroke lesions have been recognized as results of stroke. However, recent studies have suggested that inflammation occurs prior to stroke onset. In this study, we aimed to clarify the role of inflammation in stroke onset among stroke-prone spontaneously hypertensive rats (SHRSP). At 4 weeks of age (before stroke onset), the plasma level of IL-1β was significantly higher in SHRSP (153.0 ± 49.7 pg/ml) than in Wistar Kyoto rats (WKY) (7.7 ± 3.4 pg/ml, P < 0.001 versus SHRSP) or spontaneously hypertensive rats (SHR) (28.0 ± 9.1 pg/ml, P < 0.001 versus SHRSP) (n = 6 per strain). Stimulated IL-1β signal was also observed in cerebrovascular endothelial cells of SHRSP. Gene expressions of IL-1β, IL-1 receptors, caspase-1, and downstream genes (MCP-1 and ICAM-1), which associated with immune cell recruitment, were significantly greater in SHRSP than in WKY or SHR, coincident with greater NFκB protein levels in SHRSP compared to WKY or SHR. In addition, continuous administration of IL-1β (2 μg/day) using an osmotic pump slightly increased the incidence of stroke in SHR (P = 0.046) and significantly accelerated the onset of stroke in SHRSP (P = 0.006) compared to each control (n = 10 per group). These results suggest that a stimulated IL-1β signal might be a cause of stroke onset when concomitant with severe hypertension.

Dietary Protein, but Not Carbohydrate, Is a Primary Determinant of the Onset of Stroke in Stroke-Prone Spontaneously Hypertensive Rats

Background and Purpose— Previously, an inverse association has been found between the dietary proportion of protein or fat and incidence of intracerebral hemorrhage. A positive association has been found with respect to carbohydrate intake. To examine what changes in macronutrient intake are causative, animal studies were conducted. Methods— Stroke-prone spontaneously hypertensive rats (SHRSP) were fed diets with varying ratios of macronutrients ad libitum, and the onset of stroke was examined. Results— When 10% of calories were from fat, rats fed a high-protein/low-carbohydrate diet (55% calories from protein) had a delayed onset of stroke, whereas rats fed a low-protein/high-carbohydrate diet (5% calories from protein) had an accelerated onset of stroke. When 30% of calories were from carbohydrate, a marked delay in the onset of stroke was observed when the diet was high in protein. When 85% of calories were from carbohydrate, rats fed 7.5% of calories as protein displayed an accelerated onset of stroke. When 20% of calories were from protein, increased fat content did not affect the onset of stroke. However, with a fat-free diet, when 20% of calories were from protein, the onset of stroke was delayed, whereas when 10% of calories were from protein, the onset of stroke was accelerated. Conclusions— The amount of protein, but not of carbohydrate and fat, is a primary determinant of the onset of stroke. However, when calories from protein are relatively low in the diet (10%), fat is necessary to delay the onset of stroke in SHRSP.

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.

Differential Changes of Aorta and Carotid Vasodilation in Type 2 Diabetic GK and OLETF Rats: Paradoxical Roles of Hyperglycemia and Insulin

We investigated large vessel function in lean Goto-Kakizaki diabetic rats (GK) and Otsuka Long-Evans Tokushima Fatty diabetic rats (OLETF) with possible roles of hyperglycemia/hyperosmolarity and insulin. Both young and old GK showed marked hyperglycemia with normal insulin level and well-preserved endothelium-dependent and endothelium-independent vasodilation in aorta and carotid artery. There were significant elevations in endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive heme oxygenase (HO-1/HO-2) in GK. The endothelium-dependent vasodilation in GK was inhibited partly by NOS blockade and completely by simultaneous blocking of HO and NOS. In contrast, OLETF showed hyperinsulinemia and mild hyperglycemia but significant endothelium dysfunction beginning at early ages with concomitantly reduced eNOS. Insulin injection corrected hyperglycemia in GK but induced endothelium dysfunction and intima hyperplasia. Hyperglycemia/hyperosmolarity in vitro enhanced vessel eNOS/HO. We suggest that hyperinsulinemia plays a role in endothelium dysfunction in obese diabetic OLETF, while hyperglycemia/hyperosmolarity-induced eNOS/HO upregulation participates in the adaptation of endothelium function in lean diabetic GK.

Gene expression in the adrenal glands of three spontaneously hypertensive rat substrains.

We examined gene expression profiles in rat adrenal glands using genome-wide microarray technology. Gene expression levels were determined in four rat strains, including one normotensive strain [Wistar-Kyoto (WKY)] and three substrains derived from WKY rats: spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP) and malignant SHRSP (M-SHRSP). This study represents the first attempt at using microarrays to compare gene expression profiles in SHR, SHRSP and M-SHRSP adrenal glands, employing WKY as controls. Expression measurements were made in these four rat strains at 6 and 9 weeks of age; 6 weeks of age covers the pre-hypertensive period in SHR and SHRSP, and 9 weeks of age is the period of rapidly rising blood pressure (BP). Since the aim of this study was to identify candidate genes involved in the genesis of hypertension in the SHR substrains, we identified genes that were consistently different in their expression, isolating 87 up-regulated genes showing a more than 4-fold increase and 128 down-regulated genes showing a less than 1/4-fold decrease in at least two different experiments. We classified all these up- or down-regulated genes by their expression profiles, and searched for candidate genes. At 6 weeks of age, several BP-regulating genes including sparc/osteonectin (Spock2), kynureninase (Kynu), regulator of G-protein signaling 2 (Rgs2) and gap junction protein α1 (Gja1) were identified as up-regulated, and urotensin 2 (Uts2), cytoplasmic epoxide hydrolase 2 (Ephx2), apelin (Apln), insulin-like growth factor 1 receptor (Igf1r) and angiotensin II receptor-associated protein (Agtrap) were identified as down-regulated. The Kynu and Ephx2 genes have previously been reported by other groups to be responsible for hypertension in SHR; however, our present approach identified at least seven new candidate genes.

Delay of Stroke Onset by Milk Proteins in Stroke-Prone Spontaneously Hypertensive Rats

Background and Purpose— There is an inverse association between dairy food consumption and the incidence of stroke in observational studies. However, it is unknown whether the relationship is causal or, if so, what components in milk are responsible for reducing the incidence of stroke. Methods— Stroke-prone spontaneously hypertensive rats were fed diets comprising amino acids, proteins from different sources (casein, whey, soybean, or egg white), or fats from different sources (butter, beef tallow, or cocoa butter) and the onset of stroke and lifespan were examined. Results— Increasing the amount of dietary casein (5% to 55% of caloric intake) markedly delayed the onset of stroke. However, when stroke-prone spontaneously hypertensive rats were fed diets containing 55% of caloric intake as protein, rats fed casein or whey protein, a major component of milk, displayed a delayed onset of stroke compared with rats fed soybean or egg white protein. Rats fed an amino acids diet containing the same amino acids composition as casein did not have a delay in the onset of stroke. Increasing dietary fats, including butter as well as beef tallow and cocoa butter, did not affect the onset of stroke. All diets did not affect blood pressure in the early stage. Conclusions— These data suggest that the inverse association between dairy food consumption and incidence of stroke in epidemiological studies is causal and that peptides in milk protein, but not fat, might be responsible for this effect.

Continual Gram-negative bacterial challenge accelerates stroke onset in stroke-prone spontaneously hypertensive rats.

This study examined the effects of continual Gram-negative bacterial challenge on stroke onset. Stroke onset occurred significantly earlier in stroke-prone spontaneously hypertensive rats (SHRSP) injected with a bacterial cell suspension of Gram-negative rods or lipopolysaccharides (LPSs) than in uninjected controls. Paralysis of the hindlimb, piloerection, hypokinesis, and hyperkinesis were observed in LPS-injected SHRSP but not in uninjected controls during stroke onset. The serum levels of NOx, thiobarbituric acid reactive substance, and 8-hydroxydeoxyguanosine increased in LPS-injected SHRSP. These results suggest that continual Gram-negative bacterial challenge induces accelerated stroke onset in SHRSP, probably caused by oxidative stress responses derived from LPSs.

Suppression of the descending inhibitory pathway by continuous thoracic intrathecal lidocaine infusion reduces the thermal threshold of the tail-flick response in rats

PurposeFor the suppression of descending inhibitory pathways in animals, single-dose lidocaine blockade is reversible and causes less damage than chronic spinal cord injury, decerebration, and cold blockade of the spinal cord. However, single-dose blockade has a variable onset and is relatively short-lived. To surmount these disadvantages, we devised a continuous thoracic intrathecal lidocaine infusion and evaluated its effects in rats.MethodsRats were administered continuous intrathecal infusions of 0, 0.25%, 0.5%, and 1% lidocaine at 10 &gml·h−1 following a 10-&gml bolus. The effects of the continuous thoracic blockade on tail-flick (TF) latency (estimated by the percent maximum possible effect [%MPE]) and on the release of neurotransmitters in the cerebrospinal fluid (CSF) were evaluated.ResultsContinuous thoracic blockade with 0.5% and 1% lidocaine infusion reversibly shortened TF latency (%MPE, −22.0 ± 11.0 % and −21.2 ± 4.6 %, respectively, versus baseline; P < 0.05) during drug infusion. Compared with normal saline, thoracic intrathecal infusion of lidocaine significantly lowered norepinephrine and serotonin concentrations in the CSF at 1 h of infusion (P = 0.02 for both).ConclusionContinuous thoracic blockade by local anesthetic resulted in reversible suppression of descending inhibitory pathways for varying durations. Such blockade may provide further information regarding nociceptive transmission and the mechanisms of antinociception in animals.