The distribution of calbindinD-28k and parvalbumin immunoreactive neurons in the somatosensory area of the pigeon pallium.

GABAergic interneurons regulate the degree of glutamatergic excitation and output of projection neurons. In this study, we investigated the distribution of calbindinD-28k (CB) and parvalbumin (PV) in the somatosensory area of the pigeon pallium using immunohistochemical method. Our results show that anatomical structures of the somatosensory area of the pigeon pallium consisted of several subdivisions including the hyperpallium, intercalated hyperpallium, mesopallium, nidopallium and basorostralis. Neuronal density was significantly higher in the intercalated hyperpallium and basorostralis than that in the other subdivisions. The density of the CB immunoreactive neurons was generally similar in all the subdivisions; however, the density of PV immunoreactive neurons was particularly prominent in the basorostralis compared with that in the other subdivisions. In addition, the mean proportion of PV immunoreactive neurons to total neurons was higher than that in the CB immunoreactive neurons in all the subdivisions. In brief, our present study shows that PV immunoreactive neurons in the somatosensory area of the pigeon pallium were significantly abundant compared with CB immunoreactive neurons. This finding needs more studies regarding CB- and PV-related functions in the somatosensory area of the avian pallium.

The location of projection neurons to the biceps brachii muscle in the telencephalon of the pigeon.

Few studies regarding the anatomical distribution of motor neurons innervating muscles of the arm have been demonstrated in avian brains. The purpose of this study was to finely determine the localization of cerebral neurons innervating the biceps brachii muscle in the pigeon. The cholera toxin B subunit (CTB) was employed as a retrograde tracer to determine the location of neurons controlling the biceps brachii muscle in the telencephalon following intramuscular injection in male pigeons (n = 7), which were killed 14 days after intramuscular injection with CTB. We found that CTB-labelled neurons were located contralaterally in the hyperpallium apicale of the rostral telencephalon and that most of the CTB-labelled neurons were pyramidal in shape. This study shows that CTB is easily taken up by nerve terminals which innervate the biceps brachii muscle of the pigeon and that cerebral motor neurons controlling the biceps brachii muscle are located in the hyperpallium apicale.

Pituitary adenylate cyclase-activating polypeptide-immunoreactive cells in the ageing gerbil hippocampus.

In the present study, we investigated age-related changes in pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity and its protein levels in the gerbil hippocampus at various ages using immunohistochemistry and western blot analysis. In the post-natal month 1 (PM 1) group, PACAP-immunoreactive cells were found in all hippocampal subregions. The number of PACAP-immunoreactive cells was decreased in the PM 3 group and was still more decreased in the PM 6 and 12 groups. Thereafter, in the PM 18 and 24 groups, PACAP-immunoreactive cells were significantly increased again. However, in the mossy fibre zone, PACAP immunostaining was very strong in the adult group, especially in the PM 6 group. In addition, PACAP protein level was highest at PM 6, showing a slight decrease at PM 24. These results indicate that PACAP-immunoreactive cells are lowest in the adult stage and highest in the aged stage. However, PACAP immunoreactivity in the mossy fibre zone and PACAP protein level in the hippocampus are highest in the adult stage.

Microtubule associated protein 2 and choline acetyltransferase immunoreactivity in the lumbar spinal cord of young adult and aged dogs.

German Shepherds are a good model for research about aging and neurological disorders such as lumbosacral spinal canal stenosis. We compared neurons, glia and cholinergic neurons in the ventral horn of the lumbar spinal cord (L(3)) between adult (1-2 years old) and aged (10-12 years old) groups. Any pathological findings were not found by hematoxylin and eosin staining and neurological examination, and the number of NeuN (a marker for neurons)-positive neurons were similar in both groups. Microtubule-associated protein 2 (MAP2) immunoreactive dendrites in the aged dog were decreased without any change in ß-tubulin protein level. Glial fibrillary acidic protein (a marker for astrocytes) and ionized calcium-binding adapter molecule 1 (a marker for microglia) immunoreactivity were not significantly changed in both groups. The number of ChAT immunoreactive neurons was decreased; however, its protein level was not significantly changed in the aged group. These results suggest that numbers of ventral horn neurons are not changed, but cholinergic neurons may change in aged dogs compared to adult dogs.

Immunoreactivity and protein levels of olfactory marker protein and tyrosine hydroxylase are not changed in the dog main olfactory bulb during normal ageing.

The immunoreactivity and protein expression of olfactory marker protein (OMP) and tyrosine hydroxylase (TH) in the main olfactory bulb (MOB) of the dog during normal ageing was investigated. OMP immunolabelling was observed only in nerve bundles of the olfactory nerve (ONL) and glomerular layers (GL) and there was no OMP expression within cell bodies of any layer. TH immunolabelling was detected in all layers of the MOB except for the ONL. Most of the neurons expressing TH were distributed in the juxtaglomerular region and had a morphological appearance consistent with periglomerular, external tufted or superficial short axon cells. Dendrites of TH-immunoreactive neurons were closely apposed to OMP-immunoreactive nerve bundles within the glomeruli. There was no significant age-related loss of OMP and TH immunoreactivity and protein concentrations of these molecules were consistent in dogs of different ages. These results suggest that olfactory signal transduction to the GL via axons of olfactory receptor neurons remains unchanged during ageing in the dog.

The time-dependent effect of lipopolysaccharide on kainic acid-induced neuronal death in hippocampal CA3 region: possible involvement of cytokines via glucocorticoid.

It has been reported that glucocorticoid (Gc) can induce neuronal cell toxicity in the hippocampus. In addition, we examined that serum Gc increased by restraint stress aggravated kainic acid (KA)-induced neuronal death in hippocampal CA3 region. However, the effect of other stressful stimulus like lipopolysaccharide (LPS) increasing serum Gc on KA-induced neuronal death was not elucidated until now. Thus, we examined the time course effect of LPS on KA-induced neuronal death in the hippocampal CA3 region of mice, especially to address the role of Gc and inflammatory mediators. In the present study, we found that an aggravating effect of LPS on KA-induced neuronal death was correlated with an alteration of hippocampal IL-1beta mRNA level at all time points, and the serum Gc and hippocampal IL-1beta mRNA level was peak at 90 min after LPS treatment (LPS 90 min) when the aggravating effect of LPS on KA-induced neuronal death was maximum. In addition, RU38486 (glucocorticoid receptor antagonist) decreased the hippocampal IL-1beta mRNA level and abolished the aggravating effect of LPS on KA-induced neuronal death at LPS 90 min and 24 h. In the immunohistochemical study, we found activated and ramified microglia (OX-42) and astrocyte (GFAP) at 24 h after LPS treatment (LPS 24 h) in the hippocampus. These results suggest that Gc itself, cytokines triggered by Gc, or both appears to be involved in the LPS effect depending on LPS pretreatment time.

Protein phosphatase 1 nuclear targeting subunit is a hypoxia inducible gene: its role in post-translational modification of p53 and MDM2.

p53, the most commonly mutated tumor suppressor gene in human cancers, is a master regulator of apoptosis in many types of cells. Recently, protein phosphatase-1 (PP1) has emerged as a key phosphatase of p53, which modulates the interaction of p53 with its regulatory protein mouse double minute 2 (MDM2) and transcriptional activity. In the present study, we demonstrate the potential role of PP1 nuclear targeting subunit (PNUTS) in regulating the phosphorylation and apoptotic activities of p53. Hypoxia significantly increased mRNA and protein expression of PNUTS in various cell lines concomitantly with increases in p53. Promoter analysis confirmed the presence of hypoxia response elements in the promoter region of the PNUTS gene, which respond to hypoxia and forced expression of hypoxia-inducible factor 1 alpha. Overexpression of PNUTS markedly increased cell death in response to hypoxia, with increased expression of Bax, an apoptosis-related gene induced by p53. Consistently, PNUTS increased the nuclear localization, phosphorylation, and transcriptional activity of p53 as well as the ubiquitin-dependent proteosomal degradation of MDM2. However, the W401A mutant form of PNUTS, which is incapable of binding to PP1, failed to induce these events. Taken together, our findings suggest that PNUTS may play an important role in controlling cell death in response to cellular stresses such as hypoxia through the post-translational modification of p53 and MDM2.

An extract of Polygonum multiflorum protects against free radical damage induced by ultraviolet B irradiation of the skin.

Over the last decades, the incidence of ultraviolet B (UVB)-related skin problems has been increasing. Damages induced by UVB radiation are related to mutations that occur as a result of direct DNA damage and/or the production of reactive oxygen species. We investigated the anti-oxidant effects of a Polygonum multiflorum thumb extract against skin damage induced by UVB irradiation. Female SKH-1 hairless mice were divided into three groups: control (N = 7), distilled water- (N = 10), and P. multiflorum extract-treated (PM, N = 10) groups. The PM (10 g) was extracted with 100 mL distilled water, cryo-dried and 9.8 g was obtained. The animals received a topical application of 500 microL distilled water or PM extract (1, 2, 4, 8, and 16%, w/v, dissolved in distilled water) for 30 min after UVB irradiation (wavelength 280-320 nm, 300 mJ/cm(2); 3 min) of the dorsal kin for 14 days, and skin immunohistochemistry and Cu,Zn-superoxide dismutase (SOD1) activity were determined. SOD1 immunoreactivity, its protein levels and activities in the skin were significantly reduced by 70% in the distilled water-treated group after UVB irradiation compared to control. However, in the PM extract-treated groups, SOD1 immunoreactivity and its protein and activity levels increased in a dose-dependent manner (1-16%, w/v, PM extract) compared to the distilled water-treated group. SOD1 protein levels and activities in the groups treated with 8 and 16%, w/v, PM extract recovered to 80-90% of the control group levels after UVB. These results suggest that PM extract strongly inhibits the destruction of SOD1 by UV radiation and probably contains anti-skin photoaging agents.

An extract of Polygonum multiflorum protects against free radical damage induced by ultraviolet B irradiation of the skin

Over the last decades, the incidence of ultraviolet B (UVB)-related skin problems has been increasing. Damages induced by UVB radiation are related to mutations that occur as a result of direct DNA damage and/or the production of reactive oxygen species. We investigated the anti-oxidant effects of a Polygonum multiflorum thumb extract against skin damage induced by UVB irradiation. Female SKH-1 hairless mice were divided into three groups: control (N = 7), distilled water- (N = 10), and P. multiflorum extract-treated (PM, N = 10) groups. The PM (10 g) was extracted with 100 mL distilled water, cryo-dried and 9.8 g was obtained. The animals received a topical application of 500 æL distilled water or PM extract (1, 2, 4, 8, and 16 percent, w/v, dissolved in distilled water) for 30 min after UVB irradiation (wavelength 280-320 nm, 300 mJ/cm²; 3 min) of the dorsal kin for 14 days, and skin immunohistochemistry and Cu,Zn-superoxide dismutase (SOD1) activity were determined. SOD1 immunoreactivity, its protein levels and activities in the skin were significantly reduced by 70 percent in the distilled water-treated group after UVB irradiation compared to control. However, in the PM extract-treated groups, SOD1 immunoreactivity and its protein and activity levels increased in a dose-dependent manner (1-16 percent, w/v, PM extract) compared to the distilled water-treated group. SOD1 protein levels and activities in the groups treated with 8 and 16 percent, w/v, PM extract recovered to 80-90 percent of the control group levels after UVB. These results suggest that PM extract strongly inhibits the destruction of SOD1 by UV radiation and probably contains anti-skin photoaging agents.

Transient ischaemia affects plasma membrane glutamate transporter, not vesicular glutamate transporter, expressions in the gerbil hippocampus.

In the present study, we investigated expressions of vesicular glutamate transporter (VGLUT) and of the plasma membrane glutamate transporters [glutamate transporter 1 (GLT-1), glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC-1)] in the gerbil hippocampus following transient ischaemia. The expressional levels and distribution patterns of VGLUT immunoreactivities were unaltered until 3 days after ischaemic-insults. However, VGLUT-2 immunoreactivity in the CA1 region was reduced at 4 days after ischaemia due to delayed neuronal death. In addition, both GLT-1 and GLAST immunoreactivities in the CA1 region were enhanced at 30 min - 12 h after ischaemia-reperfusion and their expression began to reduce at 24 h after ischaemia-reperfusion. In contrast, EAAC-1 immunoreactivity was transiently reduced in the CA1 region at 30 min after ischaemia, re-enhanced at 3-12 h after ischaemia, and re-reduced at 24 h after ischaemia. These findings suggest that malfunctions of plasma membrane glutamate transporters, not of VGLUT, may play an important role in the elevation of extracellular glutamate concentration following ischaemic insults.

Immunohistochemical localization of glutamate in the gerbil main olfactory bulb using an antiserum directed against glutamate.

Information on the localization and the roles of glutamate in the nervous system is becoming valuable because the axon terminals of the olfactory sensory neurons and the synapses of the mitral and tufted output cells appear to be glutamatergic. In this study, we have analysed the distribution of glutamate immunoreactivity in the main olfactory bulb (MOB) of the Mongolian gerbil using an antiserum directed against glutamate. Glutamate immunoreactivity in the MOB was present in the olfactory nerve layer (Onl), glomerular layer (GL), external plexiform layer (EPL) and mitral cell layer (ML), but not in the granule cell layer (GCL). Glutamate immunoreactivity detected in the Onl was thought to be terminal ramifications of glomeruli. Some neurons in the periglomerular region showed glutamate immunoreactivity. In the EPL, glutamate immunoreactivity was found in some neuronal somata (tufted cells) and processes. In addition, mitral cells in the ML were labelled by the glutamate antibody. The pattern of glutamate immunoreactivity in the mitral cells was similar to that in the tufted cells. In brief, glutamate in the gerbil MOB is the neurotransmitter used by primary afferents and output neurons.

Corticotropin-releasing factor immunoreactivity increases in the cerebellar climbing fibers in the novel ataxic mutant mouse, pogo.

The ataxic pogo mouse (pogo/pogo) is a novel neurological mutant, which was derived as an inbred strain (KJR/MsKist) from a Korean wild mouse. The pathological manifestations include a difficulty in maintaining a normal posture, the failure of inter-limb coordination and an inability to walk straight. In this study, we examined the distribution of corticotropin-releasing factor (CRF) immunoreactive cerebellar climbing fibres and their projections to tyrosine hydroxylase (TH) immunoreactive Purkinje cells in the cerebellum of the pogo mutant mouse using immunohistochemistry. In the pogo/pogo mouse, a subset of climbing fibres was stained more intensely for CRF than in the control. Moreover, ataxic pogo mouse, neurons of the inferior olivary nucleus projecting climbing fibres were also more intensely stained for CRF than in the control. In the pogo/pogo mouse, TH immunoreactivity was located in the Purkinje cells, whereas no TH expression was found in the control. Double immunostaining for CRF and TH in the pogo/pogo cerebellum revealed that the distribution of TH-immunoreactive Purkinje cells corresponded to terminal fields of CRF-immunoreactive climbing fibres but not to the CRF-immunoreactive mossy fibres. Therefore, we suggest that an increase of CRF level may alter the function of targeted Purkinje cells and that it is related to the ataxic phenotype in the pogo mutant mouse.

Reduced calcium binding protein immunoreactivity induced by electroconvulsive shock indicates neuronal hyperactivity, not neuronal death or deactivation.

Calcium-binding proteins (CBPs), such as parvalbumin and calbindin D-28k, are useful markers of specific neuronal types in the CNS. In recent studies, expression of CBPs may be indicative of a deactivated neuronal state, particularly epilepsy. However, it is controversial whether altered expression of CBPs in the hippocampus practically indicate neuronal activity. Therefore, the present study was performed to investigate the extent of profiles of expression of CBPs in the rat hippocampus affected by several episodes induced by electroconvulsive shock. In the present study, following electroconvulsive shock expression of CBPs were reduced in the hippocampus in a stimulus-dependent manner, and recovered to the control level at 6 h after electroconvulsive shock. However, paired-pulse responses of the dentate gyrus were transiently impaired by electroconvulsive shock, and immediately normalized to baseline value. In addition, effects of electroconvulsive shock on expression of CBPs and paired-pulse responses were prevented by pretreatment of vigabatrin. These findings suggest that reduced expression of CBPs induced by seizure activity may be indicative of hyperactivity of CBP positive neurons, which is a practical consequence of the abnormal discharge, and that they may play an important role in regulating seizure activity.

Valproic acid reduces enhanced vesicular glutamate transporter immunoreactivities in the dentate gyrus of the seizure prone gerbil.

To elucidate the relationship between glutamatergic current and vesicular glutamate transporter (VGLUT) expressions, we performed the comparative analyses of evoked potentials and VGLUT immunoreactivities in the dentate gyrus, and its response to antiepileptic drug treatments in a gerbil model. The EPSP slope that could be evoked in seizure sensitive (SS) gerbils was significantly greater than in seizure resistant (SR) gerbils. There was also a strong trend towards the larger population spike amplitude in SS gerbils. In addition, VGLUT immunoreactivities were markedly enhanced in the dentate gyrus of SS gerbils, as compared with the SR gerbils. Following valproic acid (VPA, 30 mg/kg), the population spike amplitude and the EPSP slope in response to the stimulus were markedly reduced in the dentate gyri both of SR and of SS gerbils, although this dosage of VPA had no effect in low stimulus currents in SS gerbils. Vigabatrin (VGB) and low dosage of VPA treatment did not affect the evoked responses. Similarly, VPA treatment reduced enhanced VGLUT immunoreactivities in the dentate gyrus of SS gerbils, whilst VGB did not. These findings suggest that up-regulation of VGLUT immunoreactivities may be related to the hyperexcitability of granule cells in SS gerbils, and altered VGLUT immunoreactivity in the dentate gyrus may be independent of GABAergic transmission.

Histochemical and electron microscopic study on motor neurone degeneration following transient spinal cord ischaemia at normothermic conditions in rabbits.

This study was carried out to investigate the motor neurone degeneration in the ventral horn following transient spinal cord ischaemia at normothermic conditions in rabbits. Transient spinal cord ischaemia was induced by occlusion of the abdominal aorta underneath the left renal artery for 15 min at normothermia (38.7 degrees C). Sections at the level of L7 were examined using histochemical and electron microscopic methods. Cresyl violet-positive motor neurones began to reduce in number at 3 h after ischaemia reperfusion, and were not detectable at 48 h after ischaemia reperfusion. Acid fuchsin-positive motor neurones were detected at 1 h after ischaemia reperfusion, significantly increased up to 6 h after the ischaemia reperfusion, and eventually disappeared by 48 h after ischaemia reperfusion. In electron microscopic findings, the disintegration of cytoplasmic membranes, and the disruption of mitochondria and endoplasmic reticulum were observed in motor neurones at 30 min after ischaemia reperfusion. Motor neurones showed necrotic findings with pyknotic degeneration at 1 h after ischaemia reperfusion. The necrotic degeneration became severer time dependently after ischaemia reperfusion. At 48 h after ischaemia reperfusion, cellular components were not detectable in motor neurones. In conclusion, we suggest that the degeneration pattern of motor neurones of the ischaemic spinal cord was necrotic after ischaemia reperfusion under normothermic conditions.

Comparative study on calretinin immunoreactivity in gerbil and rat retina.

Expression of calretinin in retina has been ascribed to multiple biological and functional aspects in the visual system. In this study, we examined the distribution patterns of calretinin immunoreactivity in gerbil and rat retina. In the gerbil, calretinin immunoreactivity was present in bipolar and amacrine cells of the inner nuclear layer and in neurones of the ganglion cell layer. In the rat, amacrine and ganglion cells showed calretinin immunoreactivity, but bipolar cells did not contain calretinin immunoreactivity. In both species, calretinin immunoreactivity was absent in cones, cone bipolars, and horizontal cells. In conclusion, gerbil as well as rat has a rod-dominant retina. The differences in calretinin expression between rat and gerbil require further investigations under various functional and developmental conditions.

Post-natal changes of cyclin-dependent kinase 5 activator expression in the developing rat cerebellum.

cDNA of cyclin-dependent kinase 5 (Cdk5) was cloned based on its primary sequence homology to Cdc2 and Cdk2. Cdk5 requires the neuronal Cdk5 activators such as p35 or p39(nck5ai) (p39) for its activity. In this study, we examined post-natal changes in the p39 expression pattern during the development of the rat cerebellum. p39 began to express in somata and dendrites of Purkinje cells at post-natal day 3 (PD3). In particular, at PD12, parasagittal bands (stripes) with p39 immunoreactivity were weakly observed. At PD21, p39-immunoreactive stripes were developed when compared with the PD12 group. At this age stage, p39 immunoreactivity became weak in somata of Purkinje cells, not forming stripes. At PD28, a series of parasagittal bands were more distinct than those of the PD21 group, and p39 immunoreactivity disappeared in Purkinje cells, not forming p39 immunoreactive stripes. In the adults, p39 immunoreactivity in Purkinje cells was similar to that found in the PD28 group which showed that parasagittal bands were very narrow, and became progressively more slender. Therefore, we suggest that the post-natal changes of p39 expression in Purkinje cells in the cerebellum is an autonomous characteristic of Purkinje cells with a role of Cdk5 activators.

Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia.

In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5'-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1)region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12-24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway.

Changes of calbindin D-28k immunoreactivity in the hippocampus after adrenalectomy in the seizure sensitive gerbil.

Calbindin D-28k (CB), a calcium-binding protein, containing neurons in the hippocampus plays an important role in hippocampal excitability in epilepsy. In the present study, we investigated changes of CB immunoreactivity after adrenalectomy (ADX) in the hippocampus and dentate gyrus of the seizure sensitive gerbil, which is susceptible to seizure to identify roles of CB in epileptogenesis. The changes of the CB immunoreactivity after ADX were significant in the hippocampal CA1 region. By 24 h after ADX, CB-immunoreactive CA1 pyramidal cells and CB immunoreactivity increased. At this time, well-stained dendrites projected to the stratum radiatum. Thereafter, the CB immunoreactivity decreased time dependently by 96 h after ADX. In the dentate gyrus, the changes of CB-immunoreactive neurons were mainly observed in the granule cell layer. The number and immunoreactivity of CB-immunoreactive neurons was high at 24 h after ADX, thereafter, those decreased by 96 h after ADX. These results suggest that glucocorticoid has an important role in modulating the seizure activity and CB serves an inhibitory function, which regulates the seizure activity and output signals from the hippocampus.

Histochemical study on neurodegeneration in the olfactory bulb after transient forebrain ischaemia in the Mongolian gerbil.

In the present study, we investigated the ischaemia-related neurodegeneration in the main and accessory olfactory bulb (AOB) after 5 min transient forebrain ischaemia in the Mongolian gerbil using the acid fuchsin staining method. Between 5 and 15 days after ischaemia, acid fuchsin positive cells markedly increased in the external plexiform layer (EPL), mitral cell layer (ML) and glomerular layer (GL) of the main olfactory bulb (MOB), and in the mixed cell layer (MCL) and GL of the AOB. By 30 days after ischaemia reperfusion, acid fuchsin positive neurons were shrunken and showed low acidophilia in somata. Many necrotic vacuoles were found in the EPL and GL of the MOB 30 days after ischaemia. At this time, necrotic vacuoles were very few in the AOB. Therefore, our results suggest that the GL and EPL of the MOB are vulnerable to ischaemic damage at a later time after ischaemic insult, and that the AOB is more resistant to ischaemic damage as compared with the MOB.