Hisashi Ikegami

An age-related change in susceptibility of rat brain to encephalomyocarditis virus infection

Rats were inoculated intraperitoneally (i.p.) or intracerebrally (i.c.) with 1 × 104 plaque forming units (PFU)/animal of the D variant of encephalomyocarditis virus (EMC‐D) at 2, 4, 7, 14, 28 or 56 days of age for virological and histopathological examination. In the i.p.‐inoculation study, neither viral replication nor lesions were detected in the animals inoculated at 28 and 56 days of age. In the animals inoculated when younger than 14 days of age, lesions were restricted to the brain although viral replication was detected in the brain, heart and pancreas. The brain lesions were characterized by acute meningoencephalitis with neuronal necrosis in the cerebral cortex, hippocampus and thalamus, and viral RNA was detected in degenerated and/or intact neurons. In the i.c.‐inoculation study, similar age‐related changes in susceptibility of rat brain to EMC‐D infection were observed, but a minor difference was that viral replication and lesions were still detected in the hippocampus of some animals inoculated at 28 days of age. These results suggest that an age‐related decrease in the susceptibility of rat brain to EMC virus infection may reflect an age‐related change in the susceptibility of neurons themselves as well as in maturation of the immune system.

Immunohistochemical study on inducible type of nitric oxide (iNOS), basic fibroblast growth factor (bFGF) and tumor growth factor-β1 (TGF-β1) in arteritis induced in rats by fenoldopam and theophylline, vasodilators

Arteritis induced in rats by vasodilators, fenoldopam and theophylline, was examined immunohistochemically for expressions of inducible type of nitric oxide synthase (iNOS), basic fibroblast growth factor (bFGF) and tumor growth factor-beta1 (TGF-beta1). Rats were administered fenoldopam for 24 hours by intravenous infusion with or without following repeated daily oral administrations of theophylline. Irrespective of theophylline administration, iNOS antigens were remarkably abundant in ED-1-positive cells on day 5 and 8 post-fenoldopam-infusion (DPI); bFGF antigens were remarkably abundant in ED-1-positive cells on 1 and 3 DPI; TGF-beta1 antigens were observed in ED-1-positive cells on and after 5 DPI. These results suggest that the peak expression of iNOS antigen was followed by that of bFGF antigen, and bFGF may have a suppressive effect on iNOS expression in these rat arteritis models. On the other hand, TGF-beta1 was not considered to have a suppressive effect on iNOS expression in these models.

Histopathological and immunohistochemical studies on arteritis induced by fenoldopam, a vasodilator, in rats

Fenoldopam, a dopaminergic (DA1) agonist, has been reported to induce medial necrosis and adventitial inflammatory response in the splanchnic arteries in rats. This study was carried out to clarify the detailed time course of the inflammatory responses, using antibodies for the inflammatory cell markers, CD3 (T cell), CD20 (B cell) and ED-1 (macrophage), and inflammatory serum factors, IgG, IgM and C3. Rats were administered fenoldopam for 24 hours by intravenous infusion. Histopathologically, medial necrosis with hemorrhage was observed at the end of infusion, but it almost disappeared on day 7 post-infusion. Adventitial inflammatory responses with ED-1-, CD3- and CD20-positive cells were very slight at the end of infusion, became prominent with marked fibrosis on days 3 and 5, decreased on day 7, and subsided on day 14. The serum factors were first present in the area of medial necrosis, then shifted to the subendothelial space or cytoplasm of smooth muscle cells, and disappeared on day 14 post-infusion. Gaps in the external elastic lamina were observed on days 3 and 5 post-infusion, and IgG and IgM were present outside the gaps in the adventitia. These results provided us with more detailed information on the inflammatory responses following medial damage induced by vasodilators.

Enhanced nephrotoxicity of acetaminophen in fructose-induced hypertriglyceridemic rats: Effect of partial hepatectomy

Fructose-induced hypertriglyceridemic rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP). Enhanced susceptibility to APAP nephrotoxicity in fructose-pretreated rats is due, at least in part, to increased renal APAP concentration at the early phase (15 and 30 min after APAP administration). However, the mechanism of an increase in renal APAP concentration is still obscure. The present study was designed to test the hypothesis that a decrease in capacity of hepatic APAP metabolism is responsible for an increase in renal APAP concentration in fructose-pretreated rats. Non-pretreated rats and fructose-pretreated rats (25% fructose in drinking water for 3 weeks) received 70% or 90% partial hepatectomy (PH) or sham operation at 1 hr before APAP administration (600 or 750 mg/kg, i.p.). PH did not potentiate APAP nephrotoxicity and renal APAP concentration, and fructose-pretreated rats showed server renal lesions and greater renal APAP concentration than non-pretreated rats irrespective of PH. The result indicates that an increase in renal APAP concentration in the fructose-pretreated rats has no relation to an alteration in hepatic metabolic capacity of APAP.

Susceptibility of primary culture neurons from rats of different ages to encephalomyocarditis (EMC) virus infection.

The changes in susceptibility of neurons to the D variant of EMC virus (EMC-D) (10(6) PFU/well) were investigated in developing hippocampal primary cultures from postnatal days of 1, 7, and 56 Fischer 344 rats (P1, P7, and P56) for up to 12 h after infection (12 HAI). The virus titer of primary culture neurons increased at 1 HAI, decreased at 2 HAI, increased at 3 HAI, peaked at 8 HAI, and decreased at 12 HAI in all age groups. The titers at 1 and 8 HAI were lowest in P56 cultures. The virus titer of neurons was always higher than that of culture media, especially at 1 HAI, in P1 cultures, whereas the former was lower than the latter from 2 to 3 HAI in P7 cultures and from 2 to 4 HAI in P56 cultures, respectively. Signals of viral RNA detected by in situ hybridization were first observed in the peripheral cytoplasm of neurons at 1 HAI in P1 and P7 cultures and at 4 HAI in P56 cultures, respectively. The signals spread to a large or whole area of cytoplasm and also to processes thereafter. The number of viral RNA-positive neurons and the amount of signals decreased with age. The present results indicated that the susceptibility of primary culture neurons to EMC-D decreased with age but viral replication still occurred in P56 cultures.

Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats. II. Role of enhancement of fructose metabolism and overproduction of triglyceride in the liver and kidney on hepatorenal toxicity of acetaminophen

Fructose-induced hypertriglyceridemic rats are resistant to hepatoxicity and susceptible to nephrotoxicity of acetaminophen (APAP) as compared with normal ones. The present studied were designed to evaluate how fructose-treatment affects the developmental mode of hepatorenal toxicity of APAP. First, following fructose-pretreatment for various durations (1 day, 1 week or 3 weeks), 1-day-fructose-pretreatment induced hypertriglyceridemia and enhancement of APAP-nephrectoxicity simultaneously. However, it took at least 3 weeks for fructose-pretreatment to reduce APAP-hepatotoxicity. Second, following fructose, sucrose or glucose-pretreatment for 3 weeks, fructose-pretreated rats showed marked hypertriglyceridemia and modification of APAP-hepatorenal toxicity. Sucrose-pretreated rats showed less effects than fructose-pretreated rats. Glucose-pretreated rats showed no changes in plasma triglyceride and APAP-hepatorenal toxicity. Third, rats with hypertriglyceridemia induced by olive oil or Triton WR-1339 which did not produce enhanced metabolism and triglyceride-overproduction in the liver and kidney showed no modification of APAP-hepatorenal toxicity. Pretreatment of glycerol which was metabolized in liver and kidney and induced an overproduction of triglyceride resulted in an enhancement of APAP-nephrotoxicity. These results indicate that an enhancement of fructose metabolism and an overproduction of triglyceride in liver and kidney are responsible for the modification of APAP-hepatorenal toxicity in fructose-induced hypertriglyceridemic rats.

Effects of fructose-induced hypertriglyceridemia on hepatorenal toxicity of acetaminophen in rats: Role of pharmacokinetics and metabolism of acetaminophen

Fructose-induced hypertriglyceridemic rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP), as compared with normal ones. The present study was designed to test the hypothesis that alterations in the distribution of APAP and in the intrinsic susceptibility to toxicants are responsible for the alteration in hepatorenal toxicity of APAP in fructose-induced hypertriglyceridemic rats. Following APAP-administration (750 mg/kg, i.p.), fructose-pretreated rats (25% fructose in drinking water for 5 weeks) showed nephrotoxicity of APAP more promptly and more severely than normal ones. Renal APAP-concentrations at the early phase (15 and 30 min. after APAP-administration) were significantly greater in fructose-pretreated rats than those in normal ones. Plasma and hepatic APAP concentrations in fructose-pretreated rats were greater than those in normal ones only at the later phase (plasma; 6 hr, liver; 6 and 12 hr after APAP-administration). There were no significant differences in the APAP-induced depletion of hepatic and renal glutathione and in the basal hepatic and renal cytochrome P-450 contents between these rats. Fructose-pretreated rats were also more susceptible to p-aminophenol (PAP), a nephrotoxic metabolite of APAP, than normal rats. Therefore, enhanced susceptibility to APAP-nephrotoxicity in fructose-pretreated rats may be due, at least in part, to increased renal APAP concentration and increased intrinsic susceptibility to the metabolic nephrotoxicant.

Enhanced nephrotoxicity of acetaminophen in fructose-induced hypertriglyceridemic rats: contribution of oxidation and deacetylation of acetaminophen to an enhancement of nephrotoxicity.

Fructose-induced hypertriglyceridemic Sprague-Dawley (SD) rats become resistant to hepatotoxicity and susceptible to nephrotoxicity of acetaminophen (APAP) as compared with normal SD rats. Fischer-344 rats, which are susceptible to APAP nephrotoxicity, have two toxic metabolic pathways involving cytochrome P450-dependent oxidation of APAP to N-acetyl-p-benzoquinone imine (NAPQI) and P450-independent deacetylation of APAP to p-aminophenol (PAP). SD rats, however, have only the former pathway. This study was undertaken to investigate whether alterations in the metabolic pathways of APAP and in the intrinsic susceptibility to toxic metabolites are responsible for an enhancement of APAP nephrotoxicity in the fructose-pretreated SD-rats. In the non-pretreated rats, the inhibition of APAP oxidation by the MFO inhibitor, piperonyl butoxide, and deacetylation by carboxyesterase inhibitor, bis(p-nitrophenyl)phosphate, did not alter APAP-induced renal lesions. In contrast, these inhibitors protected the fructose-pretreated rats from APAP-induced renal lesions. Since there were no differences in the severity of gentamicin-, chloroform, and 45 min-ischemia/reperfusion-induced renal lesions between the non-pretreated and the fructose-pretreated rats, it is unlikely that the increased intrinsic susceptibility to chemicals and their metabolites in the fructose-pretreated rats is a major factor in the enhancement of APAP nephrotoxicity. These results indicate that the enhancement of APAP nephrotoxicity in the fructose-pretreated rats is due, at least in part, to an alteration in metabolic pathways of APAP.