Corneal lesions related to an anesthetic mixture of medetomidine, midazolam, and butorphanol treatment in rats.

An anesthetic mixture of medetomidine, midazolam and butorphanol (MMB) has been recently used in laboratory animals. We observed corneal opacity in nephrectomized rats that had undergone two operations under MMB anesthesia at 4 and 5 weeks of age. To evaluate the features of this corneal opacity, ophthalmic examinations were conducted in 83 nephrectomized rats, and 8 representative animals with corneal opacity were evaluated histopathologically 4 weeks after operation. The ophthalmic examinations revealed that 66/83 animals had corneal opacity, which was characterized histopathologically by mineralization with or without inflammation in the corneal stroma. In addition, to examine the possible causes of this corneal opacity, we investigated whether similar corneal changes were induced by the MMB anesthetic treatment in normal rats. The MMB anesthetic was administered twice to 4- and 5-week-old normal SD rats (5 animals/age) in the same manner as for the nephrectomized rats. Ophthalmic examinations were conducted in all the animals once a week, and the animals were necropsied 4 weeks after the first administration. In normal rats, similar corneal opacity was observed after the first administration, and increases in the severity and size of the corneal opacity were noted after the second administration. In conclusion, this study revealed the features of corneal opacity in rats undergoing nephrectomy under MMB anesthesia and the occurrence of similar corneal opacity in normal rats treated with MMB anesthetic. To the best of our knowledge, this is the first report of corneal opacity related to MMB anesthetic treatment in rats.

Drug-induced lenticular opacity and accumulation of cholesterol-related substances in the lens cortex of dogs.

TP0446131, developed as an antidepressant agent, was found to cause lenticular opacity in a 13-week repeated-dose study in dogs. Histopathologically, the lenticular opacity was observed as a degeneration of the lens fibers, characterized by irregularity in the ordered arrangement of the fibers which is necessary to maintain the transparency of the lens, and was considered to manifest clinically as cataract. To evaluate the development mechanism of the lenticular opacity, the chemical constituents of the lens, which is known to be associated with the development of cataract, were examined. The results of liquid chromatography-tandem mass spectrometry analysis revealed an increase in the amplitudes of 3 unknown peaks in a dose- and time-dependent manner in the lens, with no remarkable changes in the other chemical components tested. In addition, the content of cholesterol, alterations of which have been reported to be associated with cataract, remained unchanged. The mass spectral data and chromatographic behavior of the 3 peaks indicated that these peaks corresponded to sterol-related substances, and that one of them was 7-dehydrocholesterol, a precursor of cholesterol biosynthesis. This finding suggested that TP0446131 exerts some effects on the cholesterol biosynthesis pathway, which could be involved in the development of the cataracts. Furthermore, increases in the levels of these sterol-related substances were also detected in the serum, and were, in fact, noted prior to the onset of the cataract, suggesting the possibility that these substances in the serum could be used as potential safety biomarkers for predicting the onset of cataract induced by TP0446131.

Comparison of the anesthetic effect by the injection route of mixed anesthesia (medetomidine, midazolam and butorphanol) and the effect of this anesthetic agent on the respiratory function.

Recently, a mixture of medetomidine, midazolam and butorphanol (MMB) has been used as an injectable general anesthetic agent for laboratory animals. The purpose of this study was to establish data to encourage practical usage of MMB, and to clarify the effects of MMB on the respiratory function in rats. To compare the anesthetic efficacy between the injection routes, the anesthetic effects of MMB by subcutaneous (s.c.) or intraperitoneal (i.p.) injection were evaluated in rats. To assess the respiratory function, the blood gas parameters and electrolytes were assessed in serial venous blood samples collected from before s.c. injection of MMB to 270 min after the injection. Recovery from anesthesia and the respiratory changes after atipamezole injection at 30 min after MMB injection was also examined. Subcutaneous injection of MMB was associated with more rapid induction and a longer duration of anesthesia as compared to i.p. injection. The blood gas analysis findings showed MMB had effects on respiratory function, that is, elevations of the partial pressures of carbon dioxide and bicarbonate and reduction of the blood pH. Atipamezole injection resulted in recovery from the MMB-induced anesthetic effect as well as respiratory depression. In conclusion, MMB provides more effective anesthesia administered by s.c. injection compared to i.p. injection and induces respiratory change. These changes were counteracted by atipamezole. Therefore, we recommend MMB administered by s.c. injection for anesthesia, followed by injection of atipamezole after the operative procedure to allow recovery.

Selective neuronal vulnerability is involved in cerebellar lesions of Guinea pigs infected with bovine spongiform encephalopathy (BSE) prions: Immunohistochemical and electron microscopic investigations.

The cerebellar lesions of bovine spongiform encephalopathy (BSE)-infected guinea pigs were characterized as severe atrophy of the cerebellar cortex associated with the loss of granule cells, decrease in the width of the molecular layer, and intense protease-resistant prion protein (PrPSc ) accumulations that are similar to cerebellar lesions in kuru and the VV2 type of sporadic Creutzfeldt-Jakob disease. The aim of this study is to assess the relationships between the distribution and localization of PrPSc and synapses expressing neurotransmitter transporters in order to reveal the pathogenesis of the disease. We used cell-type-specific immunohistochemical makers recognizing glutamatergic and γ-aminobutylic acid (GABA)ergic terminals to identify terminals impaired with PrPSc accumulations. The distribution of PrPSc accumulations and immunoreactivity of synaptic vesicles were studied throughout the neuroanatomical pathways in cerebellar lesions. Time course study demonstrated that PrPSc accumulation showed a tendency to spread from granular layer to molecular layer. The immunoreactivity of vesicular glutamate transporter 1 (VGluT1) was localized in axon terminals of cerebellar granule cells, and decreased in association with the severity of PrPSc accumulations and loss of granule cells. Immunoreactivities of vesicular glutamate transporter 2 (VGluT2) and vesicular GABA transporter (VGAT) that exist in axon terminals of inferior olivary neurons and GABAergic synapses of Purkinje cells, respectively, were preserved well in these lesions. In brainstem, VGluT1 immunoreactivity decreased selectively in pontine nuclei that are a component of the pontocerebellar pathway, although other neurotransmitter immunoreactivities were preserved well. Our findings suggest that the selective loss of VGluT1-immunoreactive synapses subsequent to PrPSc accumulations can contribute to the pathogenesis of cerebellar lesions of BSE-infected guinea pigs.

Imaging Mass Microscopy of Kidneys from Azithromycin-Treated Rats with Phospholipidosis.

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by the excess accumulation of tissue phospholipids. Although azithromycin can be used to induce phospholipidosis, no experimental studies evaluating the relationship between drug accumulation and phospholipid localization have been performed. In this study, azithromycin was orally administered to rats for 7 days, and the relationship between drug and phospholipid accumulation was performed using imaging mass microscopy. The administration of azithromycin induced tubular epithelial vacuolation in the inner stripe of the outer medulla of the kidney, consistent with the lamellar bodies that are typical manifestations of drug-induced phospholipidosis. Azithromycin and phospholipid tissue levels were extensively elevated in the kidneys of azithromycin-treated rats. Imaging mass microscopy revealed that both azithromycin and its metabolites were found in the kidneys of azithromycin-treated rats but not in control animals. The vacuolated areas of the kidneys were primarily found in the inner stripe of the outer medulla, consistent with the areas of high azithromycin concentration. Azithromycin was colocalized with several phospholipids-phosphatidylinositol (18:0/20:4), phosphatidylethanolamine (18:0/20:4 and 16:0/20:4), and possibly didocosahexaenoyl (C22:6)-bis(monoacylglycerol) phosphate, a putative biomarker of drug-induced phospholipidosis. In summary, we found correlations between regions of kidney damage and the accumulation of azithromycin, its metabolites, and phospholipids using imaging mass microscopy. Such analyses may help reveal the mechanism and identify putative biomarkers of drug-induced phospholipidosis.

A case of spontaneous purulent granulomatous pericarditis in a beagle.

The present report describes a case of spontaneous purulent granulomatous pericarditis in a 16-month-old beagle. A gross necropsy revealed pericardial effusion and multiple nodules on the surface of the heart and around the aorta adjacent to the heart. The cut surface of these nodules was solid and white in color, containing partially yellowish white regions. Microscopically, granulomatous inflammation characterized by central necrotic cellular debris surrounded by neutrophils, macrophages, lymphocytes, plasma cells, fibroblasts and collagen fibers was observed in the epicardium. In addition, degeneration or necrosis of the arterial wall with inflammation was observed in the nodules. No gross and histological findings were observed in any organs other than the heart. Bacteria and fungi were not detected by Periodic acid-Schiff staining, Gram-Hucker staining and Ziehl-Neelsen staining. Based on these findings, the dog was diagnosed as having purulent granulomatous pericarditis. Purulent pericarditis is usually caused by pyogenic bacterial or fungus infections; however, no changes indicating a possible infection were observed in this case. In cases with spontaneous vascular changes, such as idiopathic canine polyarteritis or beagle pain syndrome, epicarditis could be secondarily caused by vascular lesions. Since this case showed different pathological features from those of spontaneous vascular changes, the pathogenesis may be different and remains unclear. To the best of our knowledge, this is the first report describing purulent pericarditis in beagles. Our case report is expected to be useful information that can be used as cardiac background findings for evaluating heart lesions in preclinical toxicology studies performed in beagles.