Silk fibroin has a protective effect against high glucose induced apoptosis in HIT-T15 cells.

High glucose levels induce cell death in many cell types, including pancreatic ß-cells. Although protective agents against glucotoxicity have been searched for extensively, so far none have been found. In this report, we tested silk fibroin (SF) as a candidate material for antiglucotoxicity in the pancreatic ß-cell (HIT-T15 cell) line. Approximately 50% of cells were killed after treatment with 80 mg/mL glucose. This reduction of cell number was recovered by the addition of SF at 50 mg/mL. SF treatment also decreased cellular reactive oxygen species (ROS) and increased proliferating cellular nuclear antigen (PCNA) immunoreactivity. In addition, TUNEL assays demonstrated that SF protects against glucose-induced apoptosis of HIT-T15 cells, suggesting that SF might protect cells from cell death by lowering cellular ROS levels. SF also induced expression of the insulin-like growth factor-1 (IGF-1) gene, and IGF-1 expression may be the cause of SF-induced protection against glucose toxicity. Taken together, these results suggest that SF could serve as a potential therapeutic agent to treat the hyperglycemia-induced death of pancreatic ß-cells.

Clusterin is highly expressed in tubular complexes during spontaneous pancreatitis of spontaneous hypertensive rats.

Pancreatitis is an inflammatory disorder of pancreas which leads to varying degrees of pancreatic endocrine and exocrine dysfunction and manifests in either acute or chronic forms. Spontaneous pancreatitis in experimental animals has rarely been reported. Here, we found acute to chronic courses of spontaneous pancreatitis in spontaneously hypertensive rats (SHRs), showing the formation of tubular complexes (TCs) and enhanced islet regeneration. We investigated the expression pattern of clusterin in the pancreas of SHRs based on immunohistochemistry (IHC). IHC analysis revealed the strong expression of clusterin in dedifferentiated duct-like cells and regenerative islets of TCs. These results imply that clusterin might be involved in the formation of TCs and parenchymal regeneration during rat pancreatitis.

Effect of aging on insulin secretory function and expression of beta cell function-related genes of islets.

Recently, the glucose-stimulated insulin release of isolated human islets has been shown to deteriorate progressively with advancing donor age. This decline in beta cell function with aging may contribute to the increasing development of IGT and type 2 diabetes and also to the progressive nature of the disease. This study was to see whether there is any change in expression of beta cell function-related genes in islets with aging. Islets were isolated from young (2-month old) and old (22-24-month old) LETO rats and C57BL/6N mice. The in vitro GSIR index was significantly lower in islets from old mice compared with young mice. In real-time RT-PCR, PDX-1, insulin, GLUT2 and prohormone convertase 1/3 gene expression in islets was markedly lower in old rats (33%, 13%, 20% and 34%, respectively) and old mice (56%, 42%, 28% and 22%, respectively) compared with young animals. On the other hand, genes not specifically related to beta cell-specific function, such as caspase 3, superoxide dismutase 2 and glycerol kinase were not significantly different in expression in islets according to age. In conclusion, with increasing age, insulin secretory function of islets deteriorates accompanied with a decrease in expression of beta cell-specific genes including PDX-1.

Transient ischemia-induced changes of excitatory amino acid carrier 1 in the ventral horn of the lumbar spinal cord in rabbits.

OBJECTIVES: To examine temporal changes of EAAC1 immunoreactivity and its protein level in the spinal ventral horn after transient ischemia in the rabbit to investigate the correlation between neuronal cell death and EAAC1 in the ventral horn of spinal cord. METHODS: White rabbits weighing 2.5-3.0 kg were anesthetized with a mixture of 2.5% isoflurane in 30% oxygen and 70% nitrous oxide, and the abdominal aortic artery below the left renal artery was occluded for 15 minutes. At designated times after reperfusion, the immunohistochemical and Western blot analysis for EAAC1 was conducted using tissues of the seventh lumbar spinal segment. RESULTS: EAAC1 immunoreactivity was detected in the neurons of the normal spinal cord. EAAC1 immunoreactivity and protein level reduced significantly 30 minutes after ischemia/reperfusion, but EAAC1 immunoreactivity and protein level again increased by 80% versus sham 3 hours after ischemia. At this time point, neurological defect in hindlimb was also detected. Thereafter, EAAC1 immunoreactivity and protein levels remained to be attenuated in the ventral horn of spinal cord until 48 hours after ischemia. CONCLUSION: The significant change in EAAC1 expression and motor defects at early time after transient spinal cord ischemia relates to the acute events following ischemia/reperfusion. These results indicate that EAAC1 has an important role in the modulation of glutamate homeostasis in ischemic neurons in the spinal ventral horn.

Age-dependent changes in iron deposition in the gerbil hippocampus.

In this study, we focused on age-dependent changes in intracellular iron deposition in the gerbil hippocampus. At 1 month of age (PM 1), iron reactivity was weak in the gerbil hippocampus. At this time, cells in the polymorphic layer of the dentate gyrus showed weak iron reactivity. At PM 3, iron reactivity in cells had not changed significantly. Thereafter, iron reactivity in the CA1-3 regions and in the dentate gyrus increased with time until PM 18. At PM 24, iron reactivity in all the subfields was similar to that at PM 18. In animals aged PM 18-24, iron positive cells had various shapes, and had processes which contained iron. These results suggest that the increase of iron deposition may be associated with normal aging and that the iron deposition in the aged hippocampus is different according to hippocampal subfields.

Immunolocalization of anion exchanger 1 (Band 3) in the renal collecting duct of the common marmoset.

The purpose of this study was to determine the expression and distribution of band 3 in the collecting duct and connecting tubules of the kidney of the marmoset monkey (Callithrix jacchus), and to establish whether band 3 is expressed in type A intercalated cells. The intracellular localization of band 3 in the different populations of intercalated cells was determined by double-labeling immunohistochemistry. Immunohistochemical microscopy demonstrated that band 3 is located in the basolateral plasma membranes of all type A intercalated cells in the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullary collecting duct (OMCD) of the marmoset. However, type B intercalated cells and non-A/non-B intercalated cells did not show band 3 labeling. Electron microscopy of the CNT, CCD and OMCD confirmed the light microscopic observation of the basolateral plasma membrane staining for band 3 in a subpopulation of interacted cells. Basolateral staining was seen on the plasma membrane and small coated vesicles in the perinuclear structure, some of which were located in the Golgi region. In addition, there was no labeling of band 3 in the mitochondria of the CNT, CCD and in OMCD cells. The intensity of the immunostaining of the basolateral membrane was less in the CNT than in the CCD and OMCD. In contrast, band 3 immunoreactivity was greater in the intracellular vesicles of the CNT. From these results, we suggest that the basolateral Cl(-)/HCO(3)(-) exchanger in the monkey kidney is in a more active state in the collecting duct than in the CNT.

The circling mouse (C57BL/6J-cir) has a 40-kilobase genomic deletion that includes the transmembrane inner ear (tmie) gene.

The circling mouse (C57BL6-cir) shows deafness and circling behavior in homozygotes. The mutation is transmitted with 100% penetrance by an autosomal recessive gene on chromosome 9. In the present study, we characterized the circling mutation as a 40-kilobase deletion that includes the transmembrane inner ear (tmie) gene. The tmie gene was first identified because its mutation causes deafness and circling behavior in spinner mice. We suggest that the genomic deletion of circling mice is a different, but allelic, mutation to that of spinner mice. In addition, during general behavioral investigations for complementation tests of the 2 strains, we found that circling and spinner mice may differ in their behavioral responses to a new environment.

Study on the ophthalmic diseases in ICR mice and BALB/c mice.

In pharmaceutical companies and research institutes, many toxicity tests are performed with laboratory animals. This study was performed to produce reference data for eye toxicity tests and to investigate the ophthalmic diseases of 408 ICR mice and 119 BALB/c mice, which are commonly used as subjects in toxicity tests. The experimental animals without clinical disorders were selected regardless of sex. The ophthalmic diseases were examined by using special ophthalmic instruments: direct ophthalmoscope, indirect ophthalmoscope, slit-lamp biomicroscope and focal illuminator. The most prevalent ocular variation within normal limits was hyaloid vessel remnant (ICR mice, 28.2%; BALB/c mice, 31.9%) and the incidence gradually decreased with age. The ocular diseases found in ICR mice were retinal degeneration (9.8%), corneal scar (4.2%), focal cataract (2.2%), anisocoria (1.2%), corneal ulcer (0.2%) and uveitis (0.2%). In BALB/c mice, corneal scar (9.2%), focal cataract (1.7%) and corneal ulcer (0.8%) were the ocular diseases found.

Age-related changes in calretinin-immunoreactive periglomerular cells in the rat main olfactory bulb.

The changes of calretinin (CR)-immunoreactive periglomerular cells in the glomerular layer of the main olfactory bulb (MOB) were investigated in rats differing ages from postnatal month 1 (PM 1) to PM 24. The number of cresyl violet-positive periglomerular cells was similar between PM 1 and PM 12, but they decreased slightly in the PM 24 group. The size of CR-immunoreactive periglomerular cells in the glomerular layer increased with age, while their numbers did not change significantly in the PM 6-PM 24 groups. In the PM 24 group, numbers of CR-positive periglomerular cell bodies and their processes decreased, while the size of CR-positive cell bodies in the glomeruli was larger than that of the previous groups. These results suggest that CR-immunoreactive periglomerular cells in the rat MOB are well-developed in the PM 6 group, and that periglomerular cells in the PM 24 group show poor CR-immunoreactivity compared to those in the PM 6 group.

Seizure-induced changes of mineralocorticoid and glucocorticoid receptors in the hippocampus in seizure sensitive gerbils.

Abnormal corticosteroid hormone levels during stress and resultant mineralocorticoid receptor (MR)/glucocorticoid receptor (GR) imbalance enhance the vulnerability of specific hippocampal neurons. In the present study, we investigated the distribution of MR and GR in seizure resistant (SR) and seizure sensitive (SS) gerbils, and observed the seizure-induced changes of MR and GR in the hippocampus of SS gerbils using immunohistochemistry and western blot analysis. MR and GR immunoreactivities were higher in the SS pre-seizure gerbils than that in SR gerbils. In the SR gerbils, the immunodensity of GR was high compared to that of MR. The changes of MR and GR immunoreactivities were significant in the stratum pyramidale of the hippocampal CA1 region and the infrablade of the dentate gyrus after seizure on-set. MR immunoreactivity in the CA1 region was significantly increased at 12h after seizure on-set, thereafter MR immunoreactivity was decreased. MR immunoreactivity in the dentate gyrus was decreased time-dependently after seizure on-set. GR immunoreactivity was decreased in the CA1 region and dentate gyrus time-dependently after seizure on-set. At 12h after seizure on-set, differences in MR and GR immunodensity diminished in the CA1 region and dentate gyrus. This imbalance of MR and GR immunoreactivity in these regions may be associated with seizure generation in the Mongolian gerbil, which is a hereditary seizure model.

The effect of P2X receptor activity on GABAA receptor-mediated inhibition in the gerbil hippocampus.

In the present study, to elucidate the effect of altered P(2)X receptor transmission on GABA(A) receptor expression and its transmission, we studied the morphological and electrophysiological responses of GABA(A) receptor in the gerbil hippocampus following P(2)X receptor antagonist/agonist treatment. Suramin or pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) treatment did not affect GABA(A) receptor immunoreactivities and paired-pulse responses in the gerbil hippocampus. In addition, ATP treatment did not significantly affect population spike amplitude ratios and EPSP slope ratios in the gerbil dentate gyrus. Co-application, but not pretreatment, of PPADS or suramin enhanced the effect of muscimol on paired-pulse inhibition in the dentate gyrus. In contrast, co-application of ATP reduced the effect of muscimol in the dentate gyrus. These findings indicate that the blockade of P(2)X receptor did not affect GABA(A) receptor immunoreactivities, and P(2)X receptor may modulate GABA(A) receptor-mediated inhibition when in co-activation with GABA(A) receptor. Therefore, our findings suggest that the relationship between GABA(A) receptor and P(2)X receptor may not be reciprocal, although GABA(A) receptor activity affects P(2)X receptor functionality and its expression.

Quantitative trait Loci for glomerulosclerosis, kidney weight and body weight in the focal glomerulosclerosis mouse model.

In 183 male progeny derived from a backcross between the FGS/Kist strain, a new mouse model for focal glomerulosclerosis (FGS) in humans, and the standard normal strain, C57BL/6J, we performed a genome-wide scan for quantitative trait loci (QTLs) affecting the glomerulosclerosis index (GSI) based on histological observation as well as kidney and body weights. Two QTLs for GSI (Gsi1-2) located on chromosomes (Chrs) 8 and 10, a kidney weight QTL (Kdw1) on Chr 19, and a body weight QTL (Bdw1) on Chr 13 were detected at the genome-wide 5% or less level. The allele derived from FGS/Kist increased GSI at Gsi1, but decreased it at Gsi2. The mice homozygous for the FGS/Kist allele decreased body and kidney weights. The identified QTLs accounted for 5-8% of the phenotypic variance.

Alterations in Na+/H+ exchanger and Na+/HCO3- cotransporter immunoreactivities within the gerbil hippocampus following seizure.

In this study, a chronological and comparative analysis of the immunoreactivities of Na(+)/H(+) exchanger 1 (NHE1), Na(+)/HCO(3)(-) cotransporter (NBC) and Na(+)/Ca(2+) exchanger (NCE) was conducted in order to identify the effects of spontaneous seizure on their protein expression levels using the gerbil model. The distribution of NHE1 and NBC immunoreactivity in the hippocampus of seizure-resistant (SR) gerbils was similar to that observed in the pre-seizure group of seizure-sensitive (SS) gerbils. From 30 min to 3 h after the onset of the seizure, both NHE1 and NBC immunoreactivities were elevated in the hippocampus, as compared to the pre-seizure group of SS gerbils. At 6 h postictal, these immunoreactivities in the hippocampus had reduced to the pre-seizure level. However, NCE immunoreactivity within the hippocampus was unaltered. These findings suggest that the changes in both NHE1 and NBC immunoreactivity within the hippocampus following seizure may affect tissue excitability and play a role in the reduction of the seizure activity in the gerbil.

The differential expression of corticotropin releasing factor and its binding protein in the gerbil hippocampal complex following seizure.

Considerable attention has been focused on the role of corticotropin releasing factor (CRF) as well as CRF-binding protein (CRF-BP) in neuropsychiatric disorders and neurodegenerative diseases including epilepsy. Therefore, in the present study, we investigated the temporal and spatial alteration of CRF and CRF-BP in the gerbil hippocampal complex in order to characterize the possible changes and associations with different sequelae of spontaneous seizure in these animals. CRF immunoreactivity was shown in the interneurons of the hippocampal complex at 30 min following seizure. Additionally, alteration of CRF-BP immunoreactivity was restricted to the entorhinal cortex after seizure. These results indicate some factors for consideration. First, in the gerbil hippocampal complex, the delayed increase of CRF immunoreactivity, in spite of its excitatory function, may attenuate seizure activity, but may not do so in epileptogenesis. Second, in contrast to the hippocampal complex, the increase in CRF-BP immunoreactivity in the entorhinal cortex following seizure may participate in feedback inhibitory modulation.

Altered corticotropin-releasing factor (CRF) receptor immunoreactivity in the gerbil hippocampal complex following spontaneous seizure.

Considerable attention has been focused on the role of corticotropin-releasing factor (CRF) in neuropsychiatric disorders and neurodegenerative diseases including epilepsy. Therefore, in the present study, we investigated the temporal and spatial alteration of CRF receptor in the gerbil hippocampal complex in order to characterize the possible changes and associations with different sequelae of spontaneous seizure in these animals. Thirty minutes postictal, a decline in CRF receptor immunoreactivity was observed in the granule cells and hilar neurons. In the subiculum, CRF receptor immunoreactivity was also significantly decreased at this time point. Twenty-four hours after seizure onset, the immunoreactivity in these regions recovered to the pre-seizure level. Moreover, 30 min after seizure in the entorhinal cortex, the density of CRF receptor immunoreactivity began to decrease, particularly in the layers II and III, compared to pre-seizure group. Nevertheless, 24h after seizure onset, CRF receptor immunodensity had recovered to its seizure-sensitive (SS) level. These results suggest that altered CRF receptor expression in the hippocampal complex may affect tissue excitability and seizure activity in SS gerbils.

Changes in Na(+)-K(+)-Cl(-) cotransporter immunoreactivity in the gerbil hippocampus following spontaneous seizure.

The immunoreactivity of Na(+)-K(+)-Cl(-) cotransporter (NKCC) in the gerbil hippocampus associated with various sequelae of spontaneous seizures were investigated in order to identify the roles of NKCC in the epileptogenesis and the recovery mechanisms in these animals. The NKCC immunoreactivities in the CA2-3 regions, the subiculum and the entorhinal cortex, were significantly more intensified in the pre-seizure group of seizure sensitive (SS) gerbils than in the seizure resistant (SR) gerbils. Following the on-set of seizure, the immunoreactivity of NKCC was significantly changed. In the hippocampal complex except the CA1 region, NKCC immunoreactivity in GABAergic neurons was significantly decreased 30 min after seizure on-set, versus the pre-seizure group. On the other hand, NKCC immunoreactivity was dramatically elevated in the CA1 regions, and 3 h postictal NKCC immunoreactivity increased significantly in the dentate gyrus and the dendrites of the pyramidal cells in the CA2-3 regions. These findings suggest that altered NKCC expression may be associated with seizure activity, and have an important role in the postictal recovery by regulating GABA-mediated inhibitory circuit in the hippocampal complex of the gerbil.

Temporal alterations in voltage gated Ca2+ channel immunoreactivities in the gerbil hippocampus following ischemic insults.

In the present study, temporal changes of voltage-gated Ca(2+) channel (VGCC) immunoreactivities were evaluated in the gerbil hippocampus following ischemia. P/Q-type VGCC immunoreactivity was elevated in the hippocampus in the 3 h post-ischemic group. In the 30 min post-ischemic group, N-type VGCC immunoreactivity began to increase only in the CA1 region. L-type (alpha1C) VGCC immunoreactivity was significantly increased in the 12 h post-ischemic group. L-type (alpha1D) VGCC immunoreactivity began to increase in the CA1 region in the 30 min post-ischemic group and peaked in the 12 h post-ischemic group. These findings suggest that the altered VGCC immunoreactivities following ischemia may play an important role in the ischemic neuronal injury.

Elevated voltage-gated Ca2+ channel immunoreactivities in the hippocampus of seizure-prone gerbil.

In present study, we investigated voltage-gated Ca2+ channel (VGCC) expressions in the hippocampus of the Mongolian gerbil and its association with different sequelae of spontaneous seizures, in an effort to identify the epileptogenesis in this animal. In the hippocampus of pre-seizure seizure sensitive (SS) gerbils, VGCC subunit expressions were significantly elevated, as compared with seizure-resistant (SR) gerbils. In 3 h postictal group, the alteration of VGCC expressions showed regional- and neuronal-specific manners; VGCC immunoreactivities in principal neurons were markedly decreased; however, their immunoreactivities in interneurons were significantly elevated. These results are the first comprehensive description of the distribution of VGCC immunoreactivities in the normal and epileptic hippocampus of gerbils, and suggest that these alterations in the hippocampus of the SS gerbil may be related with tissue excitability and have a role in modulating recurrent excitation following seizures.

Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils.

In the present study, we investigated chronological changes of galanin (GAL), well known as the potassium channel opener, immunoreactivity and GAL protein level in the hippocampus of the gerbil at the various times after 5 min transient forebrain ischemia. In the sham-operated group, weak GAL immunoreactivity was found in non-pyramidal cells. At 12 h after ischemia-reperfusion, the number of GAL-immunoreactive neurons and GAL immunoreactivity were significantly increased in the hippocampus compared to 3 h after ischemic insult, especially in the hippocampal CA1 region. Thereafter the number of GAL-immunoreactive neurons and GAL immunoreactivity decrease time-dependently in the hippocampus. Four days after transient ischemia, GAL immunoreactivity was low as compared with the sham-operated group. At this time point after ischemic insult, GAL immunoreactivity was shown in microglia in the CA1 region because delayed neuronal death happened in the CA1 pyramidal cells. The result of Western blot showed the pattern of GAL expression similar to that of immunohistochemical data. These results suggest that the early increase of GAL in the CA1 pyramidal cells may be associated with the reduction of the excitotoxic damage, that long-lasting enhanced expression of endogenous GAL at 12 h-2 days after ischemia may be associated with efflux of potassium ion into the extracellular space, and that GAL expression in microglia 4 days after ischemia may be associated with reduction of ischemic damage.

Chronological alterations of neurofilament 150 immunoreactivity in the gerbil hippocampus and dentate gyrus after transient forebrain ischemia.

In this study, we observed the chronological alterations of neurofilament 150 (NF-150) immunoreactivity in the gerbil hippocampus and dentate gyrus after 5 min transient forebrain ischemia. NF-150 immunoreactivity in the sham-operated group was mainly detected in mossy fibers and in the hilar region of the dentate gyrus. NF-150 immunoreactivity and protein contents of NF-150 and RT 97 (polyphosphorylation epitopes of neurofilament) were significantly decreased at 15 min after ischemic insult. Between 30 min and 12 h after ischemic insult, NF-150 immunoreactivity and protein content were significantly increased as compared with the sham-operated group. Thereafter, NF-150 immunoreactivity and protein content started to decrease. At 12 h after ischemic insult, unlike dentate gyrus, NF-150 immunoreactivity increased in pyramidal cells of the CA1 region. Thereafter, NF-150 immunoreactivity in the CA1 region started to decrease, and 4 days after ischemic insult, NF-150 immunoreactivity nearly was similar to that of the sham-operated group. These biphasic patterns of NF-150 immunoreactivity in the hippocampus and dentate gyrus are reverse correlated with that of the intracellular calcium influx. For calcium detection in the CA1 region, we also conducted alizarin red staining. Alizarin red positive neurons were detected in some neurons at 15-30 min after ischemic insult. At 12 h after ischemia, alizarin red positive neurons were decreased. Thereafter, alizarin red positive neurons started to decrease, but alizarin positive neurons were significantly increased in dying neurons 4 days after ischemia. These results suggest that ischemia-related changes of NF-150 expression may be caused by the calcium following transient forebrain ischemia.