Aldosterone: a mediator of retinal ganglion cell death and the potential role in the pathogenesis in normal-tension glaucoma.

Glaucoma is conventionally defined as a chronic optic neuropathy characterized by progressive loss of retinal ganglion cells (RGCs) and optic nerve fibers. Although glaucoma is often associated with elevated intraocular pressure (IOP), significant IOP reduction does not prevent progression of the disease in some glaucoma patients. Thus, exploring IOP-independent mechanisms of RGC loss is important. We describe chronic systemic administration of aldosterone and evaluate its effect on RGCs in rat. Aldosterone was administered via an osmotic minipump that was implanted subcutaneously into the mid-scapular region. Although systemic administration of aldosterone caused RGC loss associated with thinning of the retinal nerve fiber layer without elevated IOP, the other cell layers appeared to be unaffected. After chronic administration of aldosterone, RGC loss was observed at 2 weeks in the peripheral retina and at 4 weeks in the central retina. However, administration of mineralocorticoid receptor blocker prevented RGC loss. These results demonstrate aldosterone is a critical mediator of RGC loss that is independent of IOP. We believe this rat normal-tension glaucoma (NTG) animal model not only offers a powerful system for investigating the mechanism of neurodegeneration in NTG, but can also be used to develop therapies directed at IOP-independent mechanisms of RGC loss.

Expression of cytokine genes in head kidney and spleen cells of Japanese flounder (Paralichthys olivaceus) infected with Nocardia seriolae.

Nocardiosis caused by Nocardia seriolae is an important disease affecting marine fish for which neither control nor preventive measures are available. In this study, we investigated cytokine gene expression in Japanese flounder (Paralichthys olivaceus) infected with N. seriolae to understand the innate immune response. Japanese flounder were challenged with different concentrations of N. seriolae suspensions (0, 1, and 10 mg/L) by immersion for 10min. Mortality was 75% and 95% in fish infected by 1 and 10 mg/L, respectively. The expression of cytokine genes (TNF-alpha, IL-1beta, CC-chemokine) in head kidney and spleen cells to N. seriolae challenge was investigated 2, 24 h, 3 days, and 10 days post-challenge. TNF-alpha expression was significantly increased in spleen after 24 h in 1 mg/L group and in HK after 2 h in 10 mg/L group, but after 24 h and 3 days in 10 mg/L group and after 3 days in 1 mg/L group, it was significantly decreased. IL-1beta expression was significantly up-regulated in spleen after 24 h in 1 mg/l group while in HK only after 2 h in 10 mg/L group before suddenly down-regulated significantly 24 h in 10 mg/L group. The expression of CC-chemokine gene in both spleen and HK was significantly up-regulated in 10 mg/L group 2 h post-challenge and down-regulated in HK after 24 h and after 10 days in 1 mg/L group in spleen, compared to the control group.

Radial glia marker expression following experimental intracerebral hemorrhage.

In this study, we examine 3CB2 expression, a marker of radial glia, after intracerebral hemorrhage (ICH). Adult male Sprague-Dawley rats received an intracaudate injection of 100 microL autologous whole blood. Animals were sacrificed, and 3CB2 expression was quantified on Western blot. Single and double labeled immunohistochemistry was used to identify which cells express 3CB2. Neurobehavioral examinations (forelimb placing test) were perfomed as an evaluation of function. By Western blot, 3CB2 was strongly expressed at day 3 and expression persisted for at least 1 month. By immunohistochemistry, 3CB2 immunoreactivity was present in large numbers of astrocytes surrounding the hematoma at day 3 after ICH. At 1 month later, 3CB2 immunoreactivity was co-localized with a neuronal marker (TUC-4). Neurobehavioral function in the 1 month after ICH group was significantly improved compared with that of 3 days after ICH. The ICH-induced 3CB2 expression in astrocytes may reflect an early response of these cells to injury, while the delayed expression in neurons might be a part of the adaptative response to injury, perhaps leading to recovery of neurobehavioral function.

Experimental induction of nocardiosis in yellowtail, Seriola quinqueradiata Temminck and Schlegel by artificial challenge.

Challenge methods for inducing nocardiosis, caused by Nocardia seriolae in yellowtail, Seriola quinqueradiata, were evaluated. The first method involved intraperitoneal injection of 1.1 x 10(2), x 10(3) and x 10(4) cfu of N. seriolae; the second was by bath immersion with three different concentrations of bacterial suspension for 10 min; the third was by intradermal injection using a multipuncture device; the fourth was by oral administration using a tube; the fifth was based on the co-habitation of uninfected fish with others that had been artificially infected, i.e. intraperitoneally or by immersion for 10 min. The LD(50) values for the infection trials were 1.9 x 10(2) (intraperitoneal injection), 1.5 x 10(4) mL(-1) (immersion), 4.3 x 10(6) (intradermal injection) and 1.7 x 10(7) (oral administration). In the co-habitation challenge, mortalities were 70% and 50% in groups of non-infected fish mixed with fish infected by the i.p. injection and immersion methods, respectively. Fish challenged by intraperitoneal injection developed adhesions. Fish challenged by other methods did not show any gross clinical signs and moribund fish were similar to natural infection.

Detection of fish nocardiosis by loop-mediated isothermal amplification.

AIMS: Loop-mediated isothermal amplification (LAMP) is a novel method that amplifies DNA with high specificity and rapidity under isothermal conditions. In this study, using the LAMP method, a protocol for detecting Nocardia seriolae which is a causative agent of fish nocardiosis, was designed. METHODS AND RESULTS: A set of four primers, two inner and two outer, were designed based on the sequence of the 16S-23S ribosomal RNA internal transcribed spacer region of N. seriolae. Time and temperature conditions for detection of N. seriolae were optimized for 60 min at 65 degrees C. Other fish pathogen was not amplified by this LAMP system. The detection of N. seriola using LAMP was found to be more sensitive than that by polymerase chain reaction. CONCLUSIONS: LAMP is a highly sensitive and rapid diagnostic procedure for detection of N. seriolae. SIGNIFICANCE AND IMPACT OF THE STUDY: LAMP is a useful diagnostic method for fish nocardiosis.

Delayed precursor cell marker response in hippocampus following cold injury-induced brain edema.

The purpose of this study was to examine the possibility of neuronal remodeling and repair after cold injury-induced brain edema using immunoassays of nestin, 3CB2, and TUC-4. Male ddN strain mice were subjected to cold-induced cortical injury. Animals were divided into the following 6 groups: 1) 1-day after injury, 2) 1-week after injury, 3) 2-weeks after injury, 4) 1-month after injury, 5) sham, and 6) normal controls. Brain water content measurement, Western blot analysis, histological examination, and neurobehavioral examination were performed. Brain water content was significantly increased in the ipsilateral cortex at 1-day after injury. At 1-day and 1-week after injury, immunoreactivity of nestin, 3CB2, and TUC-4 were absent. Nestin was expressed in 3CB2-positive astrocytes at 1-month after injury, and nestin expression with TUC-4 was present in the hippocampal cell layer. Neurobehavioral function of the 1-month after injury group was significantly improved compared with function 1-day after injury. These results suggest that delayed precursor cell marker expression in glia and neuron-like cells might be part of adaptation to the injury. Although brain injury causes brain edema and neuronal death, there is the possibility of remodeling.

Intracerebral hemorrhage induces edema and oxidative stress and alters N-methyl-D-aspartate receptor subunits expression.

Intracerebral hemorrhage (ICH) induces brain edema formation via a variety of mechanisms including toxicity due to thrombin and erythrocyte lysis. However, the roles of oxidative damage and excitotoxicity have not been fully elucidated and they are examined in this rat ICH study. Adult male Sprague-Dawley rats received an intracaudate injection of 100 microl autologous whole blood and 5 U of thrombin. Rats were sacrificed at 1 hour, 1 and 3 days, and then the brains processed using Western blotting to quantify N-methyl-D-aspartate receptor (NR) subunit expression. At 3 days, animals were also sacrificed for assessment of protein oxidation using Western blot analysis for dinitrophenyl (DNP) and brain water content. Compared to the contralateral side, ipsilateral basal ganglia NR1 and NR2A subunit expression transiently increased at 1 hour after ICH and thrombin injection. From 24 hours there was a marked down-regulation. At 3 days, marked edema and DNP up-regulation were observed in ICH and thrombin injection groups. The present NR expression up-regulation at 1 hour may reflect the acute cell response after ICH. The down-regulation of NR subunits and upregulation of DNP may be associated with cell damage, towards which thrombin may contribute.

Post-traumatic moderate systemic hypothermia reduces TUNEL positive cells following spinal cord injury in rat.

STUDY DESIGN: A standardized animal model of contusive spinal cord injury (SCI) with incomplete paraplegia was used to test the hypothesis that moderate systemic hypothermia reduces neural cell death. Terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate [dUTP] nick-end labeling (TUNEL) staining was used as a marker of apoptosis or cell damage. OBJECTIVE: To determine whether or not moderate hypothermia could have a neuroprotective effect in neural cell death following spinal cord injury in rats. SETTING: Kagawa Medical University, Japan. METHODS: Male Sprague-Dawley (SD) rats (n=39) weighing on average 300 g (280-320 g) were used to prepare SCI models. After receiving contusive injury at T11/12, rats were killed at 24 h, 72 h, or 7 days after injury. The spinal cord was removed en bloc and of examined at five segments: 5 and 10 mm rostral to the center of injury, center of injury, and 5 and 10 mm caudal to the center of injury. Rats that received hypothermia (32 degrees C/4 h) were killed at the same time points as those that received normothermia (37 degrees C/3 h). The specimens were stained with hematoxylin and eosin, and subjected to in situ nick-end labeling (TUNEL), a specific method for visualizing cell death in the spinal cord. RESULTS: At 24 h postinjury, TUNEL positive cells (TPC) decreased significantly 10 mm rostral to center of injury in hypothermic animals compared to the normothermia group. At 72 h post-SCI, TPC also decreased significantly at 5 mm rostral, and 5 and 10 mm caudal to the lesion center compared to normothermic animals. At 7 days postinjury, a significant decrease of TPC was observed at the 5 mm rostral and 5 mm caudal sites compared to normothermic animals. CONCLUSION: These results indicate that systemic hypothermia has a neuroprotective effect following SCI by attenuating post-traumatic TPC.

Do rapid systemic changes of brain temperature have an influence on the brain?

BACKGROUND: The purpose of the present study was to examine the influence of cooling and rewarming conditions using an accurate brain temperature control system. METHOD: The brain temperature of animals was measured with a thermometer while feedback regulation was achieved with a cold (4( degrees )C) and hot (50( degrees )C) water on-off flow system. Brain temperature was well controlled throughout the experiment by using both cold water and hot water simultaneously. Three groups were studied, as follows: 1) the standard group (cooled to 24( degrees )C for 1 hour, kept at 24( degrees )C for 2 hours and rewarmed to 37( degrees )C for 1 hour), 2) the rapid-cooling group (cooled to 24( degrees )C for 30 min, kept at 24( degrees )C for 2 h, and rewarmed to 37( degrees )C for 1 h), 3) the rapid-rewarming group (cooled to 24( degrees )C for 1 h, kept at 24( degrees )C for 2 h, and rewarmed to 37( degrees )C for 30 min) and the normal-control group. FINDINGS: An increase of MAP-2 immunoreactivity of the CA1 neurons in the dorsal hippocampus was observed one week but not one month after hypothermia in the rapid-rewarming group. There was also a significant increase in the glutamate and lactate value at the end of rewarming compared with the baseline in the rapid-rewarming group (p<0.01). INTERPRETATION: Our results suggest that rapid rewarming after hypothermia triggered an uncoupling of cerebral circulation and metabolism, inducing an increase of extracellular glutamate and lactate, consequently reversible neuronal cell damage.

Embryonic intermediate filament, nestin, expression following traumatic spinal cord injury in adult rats.

Precursor cells in the ependyma of the lateral ventricles of adult mammalian brain have been reported in brain, and also in the spinal cord. The present study used antibody to the intermediate filament protein (nestin) as an immunohistochemical marker for neural stem cells and precursor cells in a rat model of spinal cord trauma. Male Sprague-Dawley rats (n=25) had a laminectomy at Thll-Thl2, and spinal cord contusion was created by compression with 30 g of force for 10 min. The rats were killed at 24 h, 1 week and 4 weeks after injury, and four levels of the spinal cord were examined: 5 mm and 10 mm, both rostral and caudal region to the injury center. Time- and region-dependent alterations of nestin immunoreactivity were analyzed. Revealed at 24 h post-injury, 5 mm rostral and caudal to the lesions, nestin expression was observed in ependymal cells and around the hemorrhagic and necrotic lesion located in dorsal spinal cord, peaking at 1 week after injury. Moreover, nestin expression was also observed in the white matter of ventral spinal cord, extending into arborizing processes centripetally from the pial surface toward the central canal. At 4 weeks after injury, nestin expression in ependyma decreased 10 mm from the injury site. But nestin expression in white matter increased dramatically with a 100-fold increase in nestin originating from the pial surface, and extension now to all the white matter. The latter was accompanied by glial fibrillary acidic protein positivity into very long arborizing processes, morphologically compatible with radial glia. The findings suggest two possible sources of precursor cells in adult mammalian spinal cord; ependyma of the central canal and subpial astrocytes. Subpial astrocytes may be associated with neural repair and regeneration after spinal cord injury.

Long-term activation of the glutamatergic system associated with N-methyl-D-aspartate receptors after postischemic hypothermia in gerbils.

OBJECTIVE: The objective of this study was to investigate whether hypothermia would suppress secondary damage in the chronic postischemic stage, in terms of glutamate excitotoxicity. METHODS: Gerbils underwent 5 minutes of ischemia via bilateral common carotid artery occlusion. Seven groups were studied, as follows: 1) ischemia without treatment group; 2) intraischemic hypothermia group; 3) postischemic hypothermia group (32 degrees C for 4 h); 4) MK-801 treatment group (2 mg/kg, every other day for 1 mo); 5) postischemic hypothermia with MK-801 treatment for 1 week group (2 mg/kg, every other day); 6) postischemic hypothermia with MK-801 treatment for 1 month group (2 mg/kg, every other day); and 7) sham-treated control group. One month after ischemia, histological changes in hippocampal CA1 neurons (assessed using hematoxylin and eosin staining) and memory function (assessed using an eight-arm radial maze) were studied. Extracellular glutamate concentrations were monitored by microdialysis during ischemia and hypothermia. Staining of microglia was performed 1 week and 1 month after ischemia. RESULTS: MK-801 alone, postischemic hypothermia alone, and postischemic hypothermia with MK-801 treatment for 1 week failed to prevent ischemic neuronal damage and memory function decreases 1 month after the insult (P < 0.05 versus control). However, the postischemic hypothermia with MK-801 treatment for 1 month group exhibited significant protective effects (not significant [P > 0.05] compared with the control group). Extracellular glutamate levels for the intraischemic hypothermia group were significantly low, compared with the postischemic hypothermia group. There was no microglial activation in the postischemic hypothermia at 1 week and 1 month after ischemia groups. CONCLUSION: Postischemic hypothermia and long-term intermittent administration of MK-801 demonstrated significant neuronal protection, indicating that long-term glutamatergic activation, with changes in N-methyl-D-aspartate receptors, plays a role in neuronal damage in the chronic postischemic stage.

Expression of an embryonic intermediate filament protein in amygdaloid kindled rats.

Amygdaloid kindling is well known as an experimental model of temporal lobe epilepsy. However, the mechanism of kindling epileptogenesis remains unclear. To examine the remodelling process in kindling, we performed immunohistochemistry of nestin, an embryonic intermediate neurofilament protein, in amygdaloid kindled rats. In rats expressing focal seizures (kindling stage C3), nestin immunoreactive cells (NIC) were detected at ipsilateral piriform cortex (PC) and ipsilateral perirhinal cortex (PRh), and at PC bilaterally in fully kindled rats expressing secondary generalized seizures (kindling stage C5). Double staining with glial fibrillary acidic protein revealed that almost all reactive astrocytes at PC express nestin immunoreactivity. These results suggest that glial NIC may participate in the remodelling process at the PC and PRh areas. This is the first report of nestin expression in kindling and suggests that glial nestin at PC and PRh may play a significant role in permanent epileptogenesis in kindling.

The contribution of low affinity NGF receptor (p75NGFR) to delayed neuronal death after ischemia in the gerbil hippocampus.

The implication of low affinity nerve growth factor receptor (p75NGFR), which is believed to play a pro-apoptotic role, in delayed neuronal death (DND) after ischemia in the gerbil hippocampus was investigated. Immunohistochemistry and Western blot analysis revealed that the presence of p75 NGFR immunoreactivity (IR) was negligible in the hippocampus of the sham control gerbil but appeared clearly in CA1 neurons 3 and 4 days after 5-min transient ischemia. Terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) positive nuclei appeared when the level of p75NGFR IR increased. Furthermore, almost all TUNEL-positive CA1 neurons also costained for p75NGFR. These results suggest that p75NGFR contributes to DND after ischemia by an apoptotic mechanism.

Collagen-binding domain of a Clostridium histolyticum collagenase exhibits a broad substrate spectrum both in vitro and in vivo.

The substrate spectrum of the tandem collagen-binding domain (CBD) of Clostridium histolyticumclass I collagenase (ColG) was examined both in vitro and in vivo. CBD bound to insoluble type I, II, III and IV collagens in vitro, and to skin, aorta, tendon, kidney, trachea and corneal tissues containing various types of collagen fibrils or sheets. CBD bound to all kinds of collagen fibrils regardless of their diameters and also bound to sheet-forming collagen in the glomerular basal lamina or Descemet's membrane of the cornea. This wide substrate spectrum expands possible applications of the drug delivery system we proposed previously (PNAS 95:7018-7023, 1998). Therapeutic agents fused with CBD will bind not only to subcutaneous tissues, but also to other tissues containing non-type I collagen.

Appearance and alteration of TUNEL positive cells through epileptogenesis in amygdaloid kindled rat.

To evaluate the role of neural cell death during and after kindling epileptogenesis, apoptotic cells were analyzed in amygdaloid kindled rat using TUNEL staining as a marker of programmed cell death. TUNEL positive cells (TPC) were stained and counted as apoptotic cells in hippocampus, white matter, diencephalon, and cortex at three stages; C0 (before kindling), C3 (during kindling) and C5 (fully kindled). The animals were studied 2 h after the last stimulation. In all regions except cortex, apoptotic cells in stage C3 were significantly increased as compared to those in stage C0. Especially, in white matter significantly more apoptotic cells in stage C3 were detected than in stage C5. The present data showed that in the course of getting the epileptogenesis, apoptosis had already occurred and this type of cell death may play a significant role in reaching stage C5 through kindling.

Depression of long term potentiation in gerbil hippocampus following postischemic hypothermia.

To investigate the mechanism of chronic cell death following postischemic hypothermia, the change of N-methyl-D-aspartate receptor (NMDAR) were examined by immunohistochemistry of NMDAR1 and long-term potentiation (LTP) in the CA1 subfield of the gerbil hippocampus. At 1 week following postischemic hypothermia (32 degrees Cx4 h), all CA1 neurons survived; however, immunoreactivity of NMDAR1 increased in neuronal perikarya whereas decreased in dendrites in the CA1 neurons. The abnormality was still observed in remaining CA1 neurons at 1 month after hypothermia. LTP was also significantly depressed at 1 week after hypothermia. These results suggest that some abnormalities in the glutamate receptor may be caused by ischemia; such abnormality would persist in spite of hypothermia treatment, resulting in the depression of LTP.

Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus.

The mechanism of neurotoxicity of Clostridium perfringens epsilon toxin to the mouse brain was investigated. Intravenous injection in mice with the toxin caused seizure and excited hippocampal neurons. Microdialysis revealed that epsilon toxin induced excessive glutamate release in the hippocampus. Both the seizure and glutamate release were attenuated by prior injection with riluzole, an inhibitor of pre-synaptic glutamate release, suggesting that this toxin enhances glutamate efflux, leading to seizure and hippocampal neuronal damage.

Identification of tranilast-binding protein as 36-kDa microfibril-associated glycoprotein by drug affinity chromatography, and its localization in human skin.

To elucidate the molecular mechanism involved in the suppression of keloids and hypertrophic scars by tranilast, we investigated the target protein of tranilast in bovine skin and aorta. A specific tranilast-binding protein was isolated from both tissues by drug affinity chromatography and was identified as 36-kDa microfibril-associated glycoprotein (36-kDa MAGP). Binding of 36-kDa MAGP to tranilast seemed to be specific since 36-kDa MAGP could be eluted from the drug affinity column by tranilast itself and also binding of 36-kDa MAGP to other anti-allergy drugs (amlexanox and cromolyn) is significantly weaker than that to tranilast. Light and electron microscopic immunohistochemistry detected the protein at the periphery of elastic fibers in normal human skin. In hypertrophic scar tissue, however, 36-kDa MAGP was located on small bundles of microfibrils. These findings provide support for the concept that elastogenesis occurs in scar tissue and 36-kDa MAGP might be one of the targets for tranilast.

Ultrastructural distribution of 36-kD microfibril-associated glycoprotein (MAGP-36) in human and bovine tissues.

We observed the ultrastructural distribution of MAGP-36 by immunoelectron microscopy in human and bovine tissues. MAGP-36 was present in microfibrils associated with tropoelastin in skin, aorta, and spleen. It was not detected in microfibrils from the ocular zonule and kidney mesangium that were not associated with tropoelastin. In skin, MAGP-36 was present in both early immature elastic fibers and mature elastic fibers. In mature elastic fibers, MAGP-36 was localized around amorphous elastic cores at the elastin-microfibril interface and in electron-dense bundles. Localization of MAGP-36 in elastic fibers coincided with the distribution of lysyl oxidase, an enzyme that plays a pivotal role in the deposition of tropoelastin. These findings suggest that MAGP-36 may be involved in elastogenesis.

Influence of rewarming conditions after hypothermia in gerbils with transient forebrain ischemia.

OBJECT: Recently, several studies have demonstrated that hypothermia has a beneficial effect on clinical outcome; however, it is difficult to determine the appropriate rewarming conditions in clinical use. The purpose of the present study was to examine the influence of rewarming conditions in gerbils with transient forebrain ischemia. METHODS: Ischemia was induced in the gerbils by a 5-minute bilateral common carotid artery occlusion, after which the animals were immediately subjected to moderate or deep hypothermia. After moderate hypothermia (30.5 degrees C for 4 hours) the animals were rewarmed over standard, fast, or slow time periods. After deep hypothermia (24 degrees C for 2 hours) the animals were rewarmed in a standard, fast, slow, or stepwise manner. Cerebral blood flow (CBF), extracellular glutamate, and lactate were monitored. Hippocampal CA I cell damage was assessed 7 days after induction of ischemia. In animals treated with moderate hypothermia, the rewarming rate had no influence on the number of surviving neurons. However, fast rewarming from deep hypothermia (to 37 degrees C for 30 minutes) failed to provide the neuroprotective effect of hypothermia. Furthermore, this group showed a poor recovery of CBF (p < 0.01) and, consequently, an increase in extracellular glutamate (p < 0.01) and lactate (p < 0.01) in the hippocampus. CONCLUSIONS: The results of this study indicate a transient uncoupling of CBF and cerebral metabolism during fast rewarming from deep hypothermia, whereas slow and stepwise rewarming periods were found to be useful for protection against uncoupling of CBF and cerebral metabolism during rewarming.