Hepatocyte growth factor incorporated into herpes simplex virus vector accelerates facial nerve regeneration after crush injury.

Hepatocyte growth factor (HGF) promotes regeneration of the central nervous system, but its effects on the peripheral nervous system remain unclear. This study was conducted to elucidate the effect of HGF on regeneration of the murine facial nerve after crush injury. To do so, a replication-defective herpes simplex virus vector that incorporated HGF was prepared (HSV-HGF). The main trunk of the facial nerve was compressed by mosquito hemostats, and HSV-HGF, control vector or medium was then applied to the compressed nerve. We found that mice in the HGF group required significantly fewer days for complete recovery from nerve compression. Furthermore, the amplitude of the evoked buccinator muscle compound action potential increased following HSV-HGF application. HGF expression in and around the compressed nerve was demonstrated by enzyme-linked immunoassay and immunohistochemistry. In addition, HSV-HGF introduction around the damaged nerve significantly accelerated recovery of function of the facial nerve. These data suggest a possible role of HGF in promoting facial nerve regeneration after nerve damage. Furthermore, this viral delivery method may be applied clinically for many types of severe facial palsy during facial nerve decompression surgery.

Neurotoxicity of 2-bromopropane and 1-bromopropane, alternative solvents for chlorofluorocarbons.

To clarify the neurotoxicity of 2-bromopropane (2-BP) in comparison with 1-bromopropane (1-BP), 36 Wistar strain male rats were divided into 4 groups of 9 and exposed daily to 100-ppm 2-BP, 1000-ppm 2-BP, 1000-ppm 1-BP, or fresh air for 8 h a day. Exposure to 1000 ppm of 1-BP was discontinued after 5 or 7 weeks' exposure because of the unexpected appearance of incomplete hindlimb paralysis followed by serious emaciation. The other groups were sacrificed at the end of 12 weeks' exposure. Exposure to 1000 ppm of 2-BP resulted in significant decreases in body weight and motor nerve conduction velocity (MCV) and elongation in distal latency (DL). A ball-like enlargement of myelin sheaths was observed. Significant reductions in the number of erythrocytes, platelets, and leukocytes, testicular germ cell loss, and seminiferous atrophy were also observed in this group, but not in 100-ppm 2-BP group. Exposure to 1000 ppm of 1-BP for 5 or 7 weeks caused a significant decrease in body weight and MCV and elongation in DL. Linearly arranged ovoid- or bubble-like debris of the axons and myelin sheaths in the teased tibial nerves and axonal swelling in gracilis nucleus were found in this group. No significant changes in hematological indices or histopathological findings of the testis were found in this group. In conclusion, 2-BP is neurotoxic to the peripheral nerves in addition to its toxic effects on the reproductive and hematopoietic systems at 1000 ppm. No noticeable changes were found in the rats exposed to 100 ppm of 2-BP. 1-BP is a potent neurotoxicant at 1000 ppm for 5 or 7 weeks, while testicular and hematopoietic toxicity was not found.

Diabetic cataract of the musk shrew (Suncus murinus, Insectivora) exhibiting spontaneous non-insulin dependent diabetes mellitus (NIDDM).

The EDS colony, developed as a new laboratory colony of the musk shrew, is characterized by a high incidence of early-onset spontaneous non-insulin dependent diabetes mellitus (NIDDM). In this colony, a few diabetic shrews exhibited a cataract at 1 month after the onset of diabetes, and all diabetic shrews had bilateral cataracts at 5 months after the onset of diabetes. In contrast, cataractous animals were never observed among non-diabetic shrews. These results suggest that the cataract in the EDS colony is a diabetic complication.

Blood and liver lipid concentrations in EDS shrews exhibiting spontaneous non-insulin dependent diabetes mellitus (NIDDM).

The EDS (early-onset diabetes in suncus) colony was developed as a new laboratory colony of the musk shrew and is characterized by a high incidence of early-onset spontaneous non-insulin dependent diabetes mellitus (NIDDM). We examined blood lipid (triglyceride [TG], total cholesterol [TC], phospholipid [PL], free fatty acid [FFA]) and liver lipid (TG, TC, PL) concentrations to investigate the features of lipid metabolism in these animals. All lipid concentrations examined both in blood and liver of the diabetic shrews had a tendency toward higher values than those in non-diabetic shrews. The PL concentration was the only parameter that barely showed a significant difference. Values for all blood lipid concentrations in diabetic shrews at 7-9 months tended to be higher than those of 2-month-old diabetic shrews, although the difference was not significant. These findings indicate that diabetic EDS shrews exhibit a much milder defect of lipid metabolism induced by NIDDM than other rodent models.

Three-dimensional observations of spatial arrangement of hepatic zonation and vein system in mice and house musk shrews.

The three-dimensional (3D) relationship among the hepatic domains and the efferent central and afferent portal veins was investigated by macroscopy, microscopy, and computer-aided 3D reconstruction methods. To clearly distinguish the pericentral domain from the periportal, we used CCl(4)-treated mice and diabetic house musk shrews, which show typical pericentral necrosis and deposition of fat, respectively. The 3D findings obtained were verified against normal control animals using advantages of our unique observations by light and fluorescent microscopy, which made it possible to differentiate the two domains well. The pericentral domains in the mice and shrews appeared three-dimensionally as continuous branched columns, and the periportal domains exist in a sponge-like network that fills the parenchymal space among the columnar pericentral domains. The efferent central veins were concentrically surrounded by the pericentral domain, and segments of the central veins flowed into large sublobular and lobar veins. The walls of these large veins faced the pericentral domain at the confluence with the central veins; the remaining portions of the walls faced the periportal domain. The afferent portal veins were placed at the two-dimensional center of the network of the periportal domain and gave off smaller portal branches radially at the intersections of the network. Three types of liver lobules-classic, portal, and acinar-have been discussed repeatedly at the (2D) level. At the 3D level, it is reasonable to consider that the liver parenchyma consists of the two continuous domains corresponding to the distribution of the vessels that we found.

Structure of the middle ear and auditory tube in the house musk shrew, Suncus murinus.

Anatomical features of the middle ear and auditory tube (AT) in the house musk shrew, Suncus murinus, were examined by dissection and light microscopy. The tensor veli palatini (TVP) and tensor tympani (TT) have no connections with the wall or cartilage of the AT although they are connected by the intermediate tendon. None of the levator veli palatini (LVP) muscle bundles are attached to the AT. The salpingopharyngeus (SA) alone has its origin on the caudal edge of the tubal cartilage. The origin extends to the pharyngeal two thirds of the cartilage. The SA originates perpendicular to the AT and runs caudomedialward. Some SA muscle bundles intermingle with those of the palatopharyngeus to end on the dorsal wall of the pharynx. The observations provide no evidence that the TVP, LVP and TT have any role in AT function. The only muscle affecting the AT function in S. murinus is the SA, and it would be the AT dilator.

Abnormal cerebral neuronal migration in a rat model of intrauterine growth retardation induced by synthetic thromboxane A(2).

Many reports have associated intrauterine growth retardation (IUGR) with adverse neurological outcome, but the underlying pathology is imperfectly understood. We have developed a new rat model of IUGR using maternal administration of synthetic thromboxane A(2) (STA(2)). In the present study, the effect of this insult on neuronal migration in the rat cerebral cortex was examined. Bromodeoxyuridine (BrdU), a time-specific cell marker was administered intraperitoneally to the mothers on embryonic day (E) 19. At postnatal day (P) 3, P4, P5, and P6, pups were terminally anesthetized and brains were removed. BrdU-labeled cells were detected immunohistochemically and counted in cerebrum, which was divided into the cortical plate (CP), the intermediate zone, and the subventricular/ventricular zone (SVZ+VZ). Numbers of labeled cells in the three areas over time were compared between IUGR and control animals. Numbers of labeled cells in SVZ+VZ were significantly greater in IUGR than in controls at P3, 5, and 6 (P<0.05). In contrast, labeled cells in the CP were significantly less abundant in IUGR animals than in controls at P3, 4, and 6 (P<0.05). We concluded that neuronal migration was delayed in IUGR rats.

1-Bromopropane, an alternative to ozone layer depleting solvents, is dose-dependently neurotoxic to rats in long-term inhalation exposure.

1-Bromopropane has been newly introduced as an alternative to ozone layer-depleting solvents. We aimed to clarify the dose-dependent effects of 1-bromopropane on the nervous system. Forty-four Wistar male rats were randomly divided into 4 groups of 11 each. The groups were exposed to 200, 400, or 800 ppm of 1-bromopropane or only fresh air 8 h per day for 12 weeks. Grip strength of forelimbs and hind limbs, maximum motor nerve conduction velocity (MCV), and distal latency (DL) of the tail nerve were measured in 9 rats of each group every 4 weeks. The other 2 rats of each group were perfused at the end of the experiment for morphological examinations. The rats of the 800-ppm group showed poor kicking and were not able to stand still on the slope. After a 12-week exposure, forelimb grip strength decreased significantly at 800 ppm and hind limb grip strength decreased significantly at both 400 and 800 ppm or after a 12-week exposure. MCV and DL of the tail nerve deteriorated significantly at 800 ppm. Ovoid or bubble-like debris of myelin sheaths was prominent in the unraveled muscular branch of the posterior tibial nerve in the 800-ppm group. Swelling of preterminal axons in the gracile nucleus increased in a dose-dependent manner. Plasma creatine phosphokinase (CPK) decreased dose-dependently with significant changes at 400 and 800 ppm. 1-Bromopropane induced weakness in the muscle strength of rat limbs and deterioration of MCV and DL in a dose-dependent manner, with morphological changes in peripheral nerve and preterminal axon in the gracile nucleus. 1-Bromopropane may be seriously neurotoxic to humans and should thus be used carefully in the workplace.

Reproductive toxicity of 1-bromopropane, a newly introduced alternative to ozone layer depleting solvents, in male rats.

1-Bromopropane has been newly introduced as an alternative to ozone-depleting solvents. We aimed to clarify its dose-dependent reproductive toxicity in male rats. Thirty-six Wistar male rats were randomly divided into 4 groups of 9. The groups were exposed to 200, 400, or 800 ppm 1-bromopropane or only fresh air, 8 h per day for 12 weeks. Epididymal sperm indices were evaluated after a 12-week exposure. The testes, epididymides, seminal vesicle, prostate, and other organs were weighed and examined histopathologically. Spermatogenic cells, in stage VII seminiferous tubules, and retained spermatids, at the basal region of stages IX-XI seminiferous epithelium, were counted. Plasma testosterone levels were measured by radioimmunoassay. The testicular weight did not significantly change, but the weight of epididymides, seminal vesicle, and prostate dose-dependently decreased. The weight of seminal vesicle decreased significantly at the lowest concentration of 200-ppm and over. 1-Bromopropane induced a dose-dependent decrease in the epididymal sperm count and in motility, as well as an increase in tailless sperm and sperm with an immature head shape. The spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, and round spermatids did not decrease significantly at stage VII. Retained, elongated spermatids near the basement membrane at the postspermiation stages IX-XI increased dose-dependently. Plasma testosterone levels significantly decreased at the 800-ppm dosage. 1-Bromopropane caused failure of spermiation. Its reproductive toxicity is different from that of 2-bromopropane, which specifically impairs spermatogonia. Thus, this solvent may have serious reproductive toxic effects in men, and should be used very cautiously in the workplace.

Fatty liver and hyperlipidemia in IDDM (insulin-dependent diabetes mellitus) of streptozotocin-treated shrews.

Severe IDDM (insulin-dependent diabetes mellitus) was produced in the musk shrew (Suncus murimus, Insectivora) by a high dose (a single intraperitoneal injection of 100 mg/kg Body Weight) of streptozotocin (STZ) injection. All shrews that were administered a high dose of STZ exhibited hyperglycemia (449 +/- 16 mg/dl vs 73 +/- 4 mg/dl in controls) and hypoinsulinemia(0.25 +/- 0.07 ng/ml vs 10.96 +/- 1.97 ng/ml in controls) with ketosuria 10 days after injection. Their livers were enlarged and exhibited ayellowish-brown color with marked triglyceride (TG) accumulation (63.25 +/- 7.10 mg/g Liver vs 2.11 +/- 0.19 mg/g Liver in controls). It is probable that the increased influx of fatty acids into the liver induced by hypoinsulinemia and the low capacity of excretion of lipoprotein secretion from liver in the musk shrew resulting from a deficiency of apolipoprotein B synthesis play important roles in fatty liver formation. Hyperlipidemia was another feature in shrews with severe IDDM. The blood TG level was especially high in these shrews (899 +/- 178 mg/dl vs 23 +/- 5 mg/dl in controls). These results indicate that the IDDM shrew, induced by high doses of STZ, is a unique model characterized by fatty liver and hyperlipidemia and may be useful for studying lipid metabolism of IDDM.

2-Bromopropane causes ovarian dysfunction by damaging primordial follicles and their oocytes in female rats.

Ovarian dysfunction induced by 2-bromopropane (2-BP) has been described in female factory workers and experimental animals. However, the underlying mechanism is still unclear. To establish the reproductive target site and define mechanisms of 2-BP toxicity in adult female rats, we examined the effects of different doses and duration of exposure to 2-BP in female rats. In the dose-dependent experiments, female rats were exposed to 2-BP at 100, 300, or 1000 ppm or fresh air (n = 9 each) in exposure chambers for 8 h/day for 9 weeks. In the time-course experiments, female rats were exposed to 2-BP at 3000 ppm for 8 h (n = 7 each). The rats were then euthanized 1, 3, 5, and 17 days after exposure. Differential follicle counts and in situ terminal deoxynucleotidyl transferase assay were used to evaluate 2-BP effect on primordial, growing, and antral follicles. Exposure to 2-BP at 300 and 1000 ppm produced a significant reduction in the percentage of primordial, growing, and antral follicles in a dose-dependent manner. Significant reduction in the percentage of primordial follicles at 17 days after exposure was observed in time-course experiments. Exposure to 2-BP at 3000 ppm for 8 h resulted in histological changes in primordial follicles complex at 5 and 17 days after exposure. These changes consisted of distortion of the symmetry of oocytes and their nuclei at Day 5 after exposure and appearance of eccentric pyknotic cells and shrinkage of oocyte nuclei at Day 17 after exposure. In situ end labeling showed increased numbers of apoptotic oocytes and granulosa cells in primordial follicles at Days 5 and 17 after exposure. Our results suggested that ovarian dysfunction induced by 2-BP was caused by the destruction of primordial follicle and its oocyte due to the induction of apoptosis. Our studies also show that the follicle differential count is a more sensitive method than the vaginal smear in monitoring the female reproductive disorders induced by 2-BP.

Effect of inhalation exposure to 2-bromopropane on the nervous system in rats.

Exposure to 2-bromopropane (2-BP) is suspected to have adverse effects on the nervous system. The aim of this study was to investigate whether the exposure of rats to 2-BP had neurotoxic effects using histological and electrophysiological studies. Wistar strain male rats were exposed daily to either 100 or 1000 ppm 2-BP or to fresh air for 8 h a day for 12 weeks. Body weight was measured before exposure and every 2 weeks. Motor nerve conduction velocity (MCV) and distal latency (DL) were measured before exposure and every 4 weeks during exposure. Histological examination of the nervous system was also performed. Exposure of rats (n = 9) to 1000 ppm resulted in suppression of body weight gain and a significant decrease in brain weight compared to the control (n = 9). Electrophysiological measurements showed a significant decrease in MCV in 1000 ppm exposed rats at 8 weeks and significant prolongation of DL at 8 and 12 weeks. Abnormalities of the myelin sheath were detected in the common peroneal nerves. In 100-ppm exposed rats (n = 9), no significant changes were noted in body weight and the peripheral nerve. In conclusions, long-term exposure to 1000 ppm of 2-BP may result in peripheral neuropathy in rats.

Protective effects of dietary cyanidin 3-O-beta-D-glucoside on liver ischemia-reperfusion injury in rats.

We recently reported that feeding cyanidin 3-O-beta-d-glucoside (C3G), a typical anthocyanin pigment, lowered the serum thiobarbituric acid-reactive substance (TBARS) concentration and increased the oxidation resistance of the serum to lipid peroxidation in rats. These results suggest that C3G acts as a potent antioxidant in vivo when acute oxidative stress is encountered. In the present study, we evaluated whether feeding C3G suppresses oxidative injury to the liver caused by hepatic ischemia-reperfusion (I/R), which was used as a model for oxidative stress. Rats were fed a diet containing C3G (2 g/kg diet) for 14 days and then subjected to hepatic I/R. I/R treatment elevated the liver TBARS concentration and the serum activities of marker enzymes (glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase) for liver injury and lowered the liver reduced glutathione concentration. Feeding C3G significantly suppressed these changes caused by hepatic I/R. Although the liver ascorbic acid concentration was also lowered by hepatic I/R, feeding C3G restored this concentration more quickly compared to the control rats. These results indicate that orally administered C3G suppresses I/R-induced oxidative damage and suggest that C3G functions as a potent antioxidant in vivo under oxidative stress.

Prevalence of herpes B virus antibody in nonhuman primates reared at the National University of Japan.

A serological investigation by means of an enzyme immuno assay test for herpes B virus (cercopithecine herpesvirus 1) was performed on 961 sera of healthy nonhuman primates reared in laboratory animal facilities which belong to the Association of Laboratory Animal Facilities of the National University of Japan. An antibody prevalence of 40% (384/ 961) was demonstrated. The antibody titer was shown to be higher among macaques (60% of cynomolgus monkeys, 53% of rhesus monkeys, and 34% of Japanese monkeys) than among non-macaque species (21%). These data indicate that nonhuman primates reared in animal facilities may present an occupational health problem and a potential zoonotic biohazard as demonstrated in limited cases in the United States.

Increased expression of 3-hydroxyanthranilate 3,4-dioxygenase gene in brain of epilepsy-prone El mice.

The El mouse is an established animal model for human epilepsy. We previously reported that the level of quinolinic acid (QUIN), an excitotoxin, was high in the brain of epilepsy-prone El mice and that the increased production of QUIN was secondary to an increased activity of 3-hydroxyanthranilate 3,4-dioxygenase (3-HAO, EC 1.13.11. 6) in the brains of these mice. In this study, we cloned and sequenced the cDNA for 3-HAO and showed that its expression in the brain of El mice was higher than that of control ddY mice. These results suggest that a genetic defect leading to derepression of the 3-HAO gene expression in the brain may be involved in the pathogenesis for the epileptic diseases of El mice.

Cream fur: a new mouse mutation that may cause unusual lipid metabolism.

In 1986 an albino mouse with cream yellow coat color was discovered in a breeding colony of strain CAL20, which is an IgH-C congenic strain of BALB/c mice, supported by the Institute for Experimental Animals, Faculty of Medicine, Kanazawa University. Genetic analysis revealed that the cream yellow coat color phenotype was controlled by a single recessive mutant gene on chromosome 13. A preliminary study with biochemical and histologic examinations showed that cream yellow hair contained more fatty acid than unaffected normal hair and a large number of lipid droplets accumulated in parenchyma cells of the liver in mutant mice, as early as the age of 32 days old, suggesting that the cream yellow coat color was due to unusual lipid metabolism. The locus was designated cream fur locus and was given the gene symbol crf. In addition, it was found that recombination frequencies in the vicinity of the region of the crf locus were markedly different between male and female meiosis.

Brainstem origin of the efferent components of the cervical vagus nerve in the house musk shrew, Suncus murinus.

The brainstem origin of the efferent neurons of the vagus nerve in the house musk shrew, an animal species which has been recently used in researches on emesis, was studied using the retrograde tracing method. The vagus nerve was exposed and cut at the mid-cervical level below the nodose ganglion. Horseradish peroxidase was applied to the proximal end of the cut nerve. The brainstem was sectioned and processed histochemically with the tetramethylbenzidine method. The horseradish peroxidase injection into the vagus nerve resulted in heavy retrograde labelling of neurons in the ipsilateral dorsal motor nucleus of the vagus nerve and ambigual nuclear complex. Labelled neurons in the dorsal motor nucleus of the vagus nerve, constituting approximately 80% of the total labelled neurons, formed a longitudinal column whose length varied from 3.4 to 3.8 mm. Half of labelled neurons in this nucleus were found at the level between the area postrema and 0.6 mm rostral to it. The ambigual nuclear complex was made up of two major longitudinal divisions; the dorsal division corresponded to the ambiguus nucleus and the ventral division was identified as the external formation of the ambiguus nucleus. Our results suggest that in the Suncus murinus the neuroanatomical feature of the dorsal motor nucleus of the vagus nerve is similar to those of other mammals, but ambigual nuclear complex must be somewhat different between mammals.

Toxin-induced IDDM (insulin dependent diabetes mellitus) in the musk shrew.

We administered streptozotocin (STZ) and alloxan (AL) to the musk shrew (Suncus murinus, Insectivora) to determine the effective diabetogenic dose of the two toxins for this species. A single intraperitoneal (i.p.) injection of 75 mg/kgBW or the consecutive 5-day s injection of 25 mg/kgBW of STZ to non-fasted shrews, effectively (100%) induced hyperglycemia (> or = 300 mg/dl) with hypoinsulinaemia (< 30% of control level) in male shrews at 10 days after administration. Morphological studies showed cytological changes of B cells in the pancreatic islets of diabetic shrews. Hyperglycemic shrews induced by STZ were thus in IDDM (insulin dependent diabetes mellitus), and showed high susceptibility to the diabetogenic effect of STZ as compared with rodents. Shrews showed a sex difference in the diabetogenic susceptibility to STZ as do mice (male > female). They also showed a species specific resistance to the diabetogenic effect of AL. Of the eight shrews (with 8-hr fasting) that has been treated with a single injection of 200 mg/kgBW of AL, seven (88%) survived at least 10 days, showing no signs of hyperglycemia. All shrews died within 3 day s after injection of 250 or 300 mg/kgBW. These results indicated that the STZ-induced diabetic shrew is a unique animal model and may be useful for IDDM research. On the other hand, the musk shrew was highly resistant to the diabetogenic effects of AL.

Brainstem topology of the vagal motoneurons projecting to the esophagus and stomach in the house musk shrew, Suncus murinus.

The central origin of vagal efferents innervating the esophagus and stomach in the house musk shrew, Suncus murinus, was studied using the retrograde tracing technique. The animals were perfused with fixative 48-72 h after HRP injection and sections were processed by HRP histochemistry. HRP application into the gastroesophagus resulted in bilateral labelling of neurons in the dorsal motor nucleus of the vagus nerve (DMX) and ambiguous nucleus (AN). Labelled neurons in the DMX were observed from all regions except from the cervical esophagus, while ones in the AN were seen from the esophagus and cardia. The more labelled neurons were observed on the right DMX from subdiaphragmatic esophagus, cardia, lesser curvature and ventral corpus, while on the left DMX from the dorsal corpus labelled neurons in the longitudinal extent of the DMX were generally located at the dorsal and dorsomedial part, and those in the middle part were scattered. Labelled neurons in the AN were located restricted in the rostral part. Our results suggest that in the Suncus murinus the rostrocaudal site-specific localization within the DMX was not found, but it was prominent in the AN. In addition, while the majority of neurons which supply the esophagus and stomach were located in the DMX, only a small number was found in the AN.

Serotonergic neurons are present and innervate blood vessels in the olfactory bulb of the laboratory shrew, Suncus murinus.

The distribution and characteristics of serotonin-immunoreactivity in the olfactory bulb of the laboratory shrew (Suncus murinus, insectivore) was studied immunohistochemically. Serotonergic neurons were found only in the subependymal layer of the main olfactory bulb. These neurons were 8-12 microm in size and bipolar in shape. These serotonergic neurons had smooth nerve fibers which innervate blood vessels located mainly in the subependymal layer of the main olfactory bulb. On the other hand, other serotonergic nerve fibers with varicosities, which must be extrinsic, were detected in most olfactory layers except the olfactory nerve layer. This result suggests that intrinsic serotonergic neurons may control blood vessels and varicose serotonergic nerve fibers may act to modulate the olfactory transmission.