Kanji Muramatsu

Vulnerability to Cerebral Hypoxic-Ischemic Insult in Neonatal but Not in Adult Rats Is in Parallel With Disruption of the Blood-Brain Barrier

BACKGROUND AND PURPOSE Vulnerability to cerebral hypoxic-ischemic (H-I) insult and its relation to disruption of the blood-brain barrier were investigated in postnatal rats. METHODS Pups of postnatal day (P) 7, P14, and P21 underwent ligation of a unilateral carotid artery and were exposed to hypoxic conditions. For the detection of early-phase deterioration, brains were perfusion-fixed 24 hours after H-I insult and examined by argyrophil III method. For the detection of later infarction, animals were fixed at 72 hours after the H-I insult. RESULTS In either case, tissue damage was detected in the striatum, parietal cortex, and hippocampus. The vulnerability of P7 and P21 rats was remarkable, as compared with P14 rats. Although the developmental status of the vasculature was not significantly different at each age, the permeability of IgG after H-I injury was prominent in P7 rats and to a lesser extent in P14 rats. In P21 rats, however, there was little IgG leakage even 24 hours after the insult. Dexamethasone pretreatment blocked the extravasation of IgG and reduced the damaged tissue in P7 and P14 rats but not in P21 rats. Percentages of reduction in infarcted areas by the dexamethasone became smaller in proportion to ages. CONCLUSIONS The results suggest that in younger rats vulnerability to H-I insult was in parallel with permeability of the blood-brain barrier, whereas in adults in might be more dependent on cellular vulnerability.

Estrogen protects against while testosterone exacerbates vulnerability of the lateral striatal artery to chemical hypoxia by 3-nitropropionic acid

Gender differences in the vulnerability of the lateral striatal artery (1STR artery) to systemic intoxication with 3-nitropropionic acid (3-NPA, succinate dehydrogenase inhibitor) were studied. Subcutaneous injection of 3-NPA (20 mg/kg once a day for 2 days) induced striatal selective lesions in half of male rats associated with motor symptoms (rolling, paddling, recumbency, etc) while female rats were resistant. Lesions were located in the lateral striata and characterized by astroglial necrotic cell death, enhanced immunoreaction to factor VIII-related antigen, edema, extravasation of IgG and sometimes bleeding. The motor and histological disturbances were highly sex-dependent and modulated by changes in hormonal levels. Males were more susceptible than females. Castration had little effect but ovariectomy enhanced the vulnerability. Replacement therapy with testosterone increased while estradiol or tamoxifen suppressed the vulnerability in ovariectomized females. Investigation of the arterial architecture of the brain often revealed rectangular and acute angled branchings in the centrolateral striatum where the ISTR artery feeds. A parallel in vitro toxicity study demonstrated that an extreme Ca++ overload and a strong cellular swelling resulted in astrocytic cell death. Data suggest that 1STR artery and astrocytes are highly vulnerable to 3-NPA intoxication in males. The greater vulnerability of the ISTR artery may contribute to the pathogenesis of neurodegenerative diseases, striatal bleeding, etc. Protective effects of estrogen and tamoxifen may mediate gender differences often observed in these disorders and suggest their potential use as therapeutic agents for these disorders.

Simultaneous optical imaging of intracellular Cl- in neurons in different layers of rat neocortical slices: advantages and limitations.

Simultaneous recording of changes in intracellular Cl- concentration ([Cl-]i) in individual neurons situated in different layers (e.g. II/III-VI) of neocortical slices was found to be feasible by means of optical fluorescence measurements using 6-methoxy-N-ethylquinolinium iodide (MEQ). Gamma-aminobutyric acid (GABA) caused a measurable increase in [Cl-]i in adult neocortical neurons, but a decrease in immature neurons. Developmental changes in the function of the Cl- pump and cation-Cl- co-transporters were evaluated using inhibitors such as furosemide (FURO), ethacrynic acid (ETA), and bumetanide (BMT). However, it was found that these inhibitors absorb and/or emit light of the wavelength that is used for the optical imaging of MEQ. In addition, quenching of MEQ fluorescence by Cl- and leakage of loaded MEQ was significantly enhanced at a higher temperature, which will limit experimentation at > 30 degrees C. Estimation of [Cl-]i in individual neurons in slices was made possible by calibrating intracellular MEQ fluorescence signals at known Cl- concentrations ([Cl-]) in the presence of tributyltin, a Cl(-)-OH- antiporter, nigericin, a K+-H+ antiporter, and KSCN. This enables comparison of [Cl-]i between neurons in different slices. Thus, optical imaging of [Cl-]i in brain slices can provide valuable spatial information about [Cl-]i dynamics and homeostasis, although it should be emphasized that the technique does have some limitations.

Optical imaging reveals cation-Cl^- cotransporter-mediated transient rapid decrease in intracellular Cl^- concentration induced by oxygen-glucose deprivation in rat neocortical slices

In brain slices from young (postnatal day (P) 10--15) rat somatosensory cortex, real-time neuronal intracellular Cl(-) concentration ([Cl(-)](i)) recordings were made by an optical technique measuring 6-methoxy-N-ethlquinolinium iodide (MEQ) fluorescence. Oxygen--glucose deprivation (in vitro model of ischemia) induced a long-lasting [Cl(-)](i) increase preceded by a rapid, transient [Cl(-)](i) decrease that could not be inhibited by blockers of Cl(-) pumps, Cl(-) channels, or Cl(-) antiporters, but was sensitive to cation-Cl(-) cotransporter inhibitors (bumetanide and furosemide). Use of low external Na(+) or high external K(+) revealed that the Na(+),K(+)-2Cl(-) cotransporter was inhibited by bumetanide and furosemide, whereas the K(+)-Cl(-) cotransporter was preferentially inhibited by furosemide under our experimental conditions. With a reduced inward driving force for Na(+) (reducing Na(+),K(+)-2Cl(-) cotransport), the transient [Cl(-)](i) decrease was only rarely induced by oxygen-glucose deprivation. In contrast, with a reduced outward driving force for K(+) (reducing K(+)-Cl(-) cotransport), the transient [Cl(-)](i) decrease still occurred. These results suggest that the transient [Cl(-)](i) decrease was primarily mediated by a rapid inhibition of the inwardly directed Na(+),K(+)-2Cl(-) cotransporter. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments suggested that the isoform involved is NKCC1. We hypothesize that the initial rapid Cl(-) efflux might effectively delay the irreversible Cl(-) influx that mediates neuronal injury.

Distribution of μ-calpain proenzyme in the brain and other neural tissues in the rat

We raised antibodies against the acetyl N-terminal peptide of the human mu-calpain 80 kDa (80 K) subunit (N-acetyl SEETPVYCT-GVSAQVQKQRARELG) in the rabbit. A specific antibody was purified using N-acetyl SEEITPVYCTGVSAQVQKQ peptide-conjugated Sepharose 4B as an affinity gel support. Epitope analysis revealed that the purified antibody reacted only with mu-calpain N-terminal peptides containing N-acetyl SEETT structure but no reactions occurred with other analogous peptides. Western blot analysis showed that the antibody reacted with both human and rat mu-calpain proenzymes but not with the activated calpains lacking N-terminal peptide. Using this antibody we investigated immunohistochemically the distribution of mu-calpain proenzyme in central and peripheral nervous systems as well as other non-neural tissues in the rat. The proenzyme was detected mainly in neurons both in the central and peripheral nervous tissues, but not in non-neural tissues except for red blood cells. Immunoreaction was stronger in the perikarya and/or in the nuclei than in-the cytoplasm. Specificity of the antibody was verified by an absorption test. In summary, the mu-calpain proenzyme is mainly distributed in the perikarya and/or nuclei or neurons. Our present antibody specific to the N-terminus of the mu-calpain 80 K subunit could serve as a useful tool to detect various functions of mu-calpain as well as the damage in neurons caused by the enzyme.

Day-night differences in the contents of vasoactive intestinal peptide, gastrin-releasing peptide and Arg-vasopressin in the suprachiasmatic nucleus of rat pups during postnatal development

To investigate the developmental features of peptide levels in the suprachiasmatic nucleus (SCN), the contents of vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP) and Arg-vasopressin (AVP) were measured in punched-out SCN tissue from rat pups on postnatal days (PD) 4, 15 and 20 using a specific enzyme immunoassay. The VIP content was high at night, and the day-night difference was largest at PD4 throughout the developmental stage. GRP and AVP contents increased following the maturation. GRP levels were higher during the dark than the light period from PD4 to PD20. Subjective day-night differences were also observed in blinded pups. Our results indicate that VIP, GRP and AVP in the SCN already show a diurnal rhythm in the SCN during the nursing period.

Biphasic changes in intracellular Cl− induced by oxygen-glucose deprivation in rat neocortical slices: A study by optical imaging

YASUMASA YAMADA’,’ ATSUO FUKUDA MASAKI TANAKA4 KANJI MURAMATSU’ HAJIME TOGARI’ HIT00 NISHINO’ AND YOSHIRO WADA’ 1Dept. Pediatr. & 2Lkpt. Physiol., Nagoya City Univ. Med. Sch., Nagoya 467-8601, 3 Dept. Physiol., Hamamatsu Univ. Sch. Med., Hamamatsu 431-3192,4 Dept. Neuropsyc., Natl. Epilepsy Ctr., Shizuoka 420-8688 GABAergic actions often become excitatory rather than inhibitory during hypoxia/ischemia in the cortex. A loss of the normal Cl gradient during hypoxia/ischemia has been suggested to be involved in the GABAergic effect. Recently simultaneous recording of intracellular Clconcentration ([Cl],) in individual neurons of neocortical slice has become feasible by means of optical fluorescence measurements using 6-methoxy N-ethylquinolinium iodide (MEQ_). This technique provides information about [Cl-], dynamics and homeostasis. When slices of somatosensory cortex of immature rats (PlO-14) were subjected to a transient ischemia in vitro (oxygen-glucose deprivation for 1Omin at 30”C),there was a slight increase followed by an abrupt decrease in MEQ fluorescense in individual neurons.These changes in MEQ fluorescense were not parallel with those of cellular volume, which was measured with a ion/pH insensitive dye, calcein-AM. This result indicates that the biphasic change in MEQ fluorescense is not an effect of condensation or dilution of the dye by the change in cellular volume,but rather suggesting an initial efflux followed by massive influx of Cl-.This imbalance in Clhomeostasis may partly be involved in pathogenesis of neonatal brain ischemia.

Differential vulnerability to cellular Ca accumulation promoted by 3-nitropropionic acid (3-NPA) in cultured neurons and astrocytes

Takuya Sakaguchi, Motoy Kuno, Kazuo Kawasaki Neuronal death induced by glutamate or other excitatory amino acids can be classified into the rapidly (<a few hrs) developing and slowly evolving forms. The rapid neuronal death has been postulated to result from osmotic uptake of water (swelling) and lysis. We applied NMDA to rat hippocampal slice cultures for 15 min, and the resultant neuronal death was quantified by propidium iodide 1 hr later. The magnitude of NMDA-induced neuronal death occurred in a dose-dependent manner, but the magnitude of cell swelling was independent of the NMDA dose applied in a range of 20-100pM. Cell swelling was also induced by application of hypotonic solutions for 15 min. The hypotonic insult failed to induce cell death even when the magnitude of cell swelling was much greater than that observed by NMDA application. It is concluded that cell swelling is no the primary

1335 Susceptibility to hypoxic-ischemic brain damagein neonatalrats is closely related with development of blood-brain barrier

Unilateral carotid artery hgations were made in postnatal day (P) 7, 14 and 21 rats, that were exposed to the hypoxic (8% oxygen) situation for 1.5 h later on. The sections from these animals were examined with argyrophil III and hematoxylineosin methods, and the vulnerability to the hypoxic-ischemic (H-I) brain damage was investigated. Unexpectedly, the histopathological damage did not proportionate to age (W=P21<P14). Generally, immature cells are believed to be resistive to ischemic situation, therefore the results might be modified by other factors specially susceptible in younger age. As a candidate, the permeability of BBB was evaluated. It was revealed by immunohistological methods that younger rats had significantly higher Pcarmcability of IgG after H-I exposure. With dexarnethasone pretreatment to block the extravasation of plasma, the reduction of damage was only apparent in younger rats and the vulnerability became proportional to age (P75 P14<P21). These results suggest that the vulnerability to H-I neuronal damage was closely related to that of BBB in the neonatal rats.