Yasunobu Shimano

GDNF is a major component of trophic activity in DA-depleted striatum for survival and neurite extension of DAergic neurons

Extracts from dopamine (DA)-depleted striatal tissue (lesion extract) and from intact striatal tissue (intact extract) were prepared, and trophic activities in these extracts were evaluated using survival and neurite extension of DAergic neurons as indices. Levels of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), glial cell-line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) in extracts were measured using enzyme-linked immunosorbent assay (ELISA). The lesion extract exhibited a stronger trophic activity on survival and neurite extension of DAergic neurons than intact extract. In lesion extract, bFGF was slightly and GDNF was significantly increased, while BDNF and NT-3 were the same level in each extract. The peak increase of bFGF and GDNF was during 2 to 3 weeks after DA depletion. Trophic activity of extract was strongly attenuated after immunoprecipitation of GDNF and partly attenuated after immunoprecipitation of bFGF. In parallel immunohistological study, no significant variations were found for striatal microtubule-associated protein-2 (MAP-2)- nor OX-41-immunoreactive cells, while the number of strongly labeled glial fibrillary acidic protein (GFAP)-immunoreactive cells were increased in DA-depleted striatum, suggesting reactive gliosis. Data suggest that bFGF is a minor, while GDNF is a major component of trophic activity for DAergic neurons in DA-depleted striatum, and increased bFGF and GDNF levels may be mediated partly by reactive gliosis.

Chronically administered 3-nitropropionic acid induces striatal lesions attributed to dysfunction of the blood-brain barrier

3-Nitropropionic acid (3-NPA), an irreversible inhibitor of succinate dehydrogenase, was administered to rats and the characteristics of the neuronal damage were investigated. Injections of 3-NPA (15 mg/kg s.c.) every 2 or 3 days for 1-2 weeks induced a mild neuronal loss and neutrophil invasions in the striatum (STR). The same administration for 4 weeks induced specific symmetric lesions in the lateral STR although the size was variable in each animal. Inside the lesions, strong neutrophil invasions and a strong immunoreaction for IgG, C3 as well as complement factor C3b/C4b receptor (C3b/C4br) were detected. Lesioned sites lost the immunoreaction for GFAP while the marginal areas contained abundant GFAP-labeled astrocytes around the vessels. In intoxicated animals, there was a weak but stout immunoreaction for IgG and C3b/C4br localizing around vessels in the STR even when there were no lesions or neuronal loss. The data suggest that the blood-brain barrier dysfunction is responsible for the specific vulnerability of the STR for the toxin.

3-Nitropropionic acid produces striatum selective lesions accompanied by iNOS expression

Systemically administered 3-nitropropionic acid (3-NPA) that inhibits the mitochondrial oxidative phosphorylation induces selective lesions in the striatum. To investigate the nature of these selective lesions, we administered 3-NPA (20 mg/kg, s.c. daily for 2 or 3 days) to Wistar rats and investigated the behavioral disturbance, striatal lesions and their variations after modulating the activity of nitric oxide synthase (NOS). On the second or third day of 3-NPA administration, half the animals manifested behavioral disturbances (paddling, rolling, tremor, abnormal gait, and recumbence). A strong extravasation of immunoglobulin G (IgG) and a decrease in immunoreaction for glial fibrillary acidic protein (GFAP) were detected, and iNOS-like (iNOS-L) immunoreactive small cells appeared in the lateral and central striatum especially around the vessels. A week later, lesions lacking GFAP-immunoreaction were detected in the striatum in survived animals. Pretreatment with N-nitro-L-arginine methyl ester (L-NAME) along with each injection of 3-NPA did not improve the behavioral disturbances nor the survival rate, but attenuated the extravasation of IgG and iNOS-L immunoreaction. Pretreatment with aminoguanidine or FK506 improved the behavioral symptoms and survival rate. Extravasation of IgG and expression of iNOS-L immunoreactivity were attenuated, and the striatal lesion was reduced. Data indicate the involvement of NO in the high vulnerability of the striatum, and that iNOS, one of inflammatory markers, is induced following exposure to 3-NPA.

Complement fragment C4d deposition in peritubular capillaries in acute humoral rejection after ABO blood group-incompatible human kidney transplantation.

Background. Acute humoral rejection (AHR) is the most important risk factor for early graft loss in ABO-incompatible (ABO-i) kidney transplantation (RTx). The pathogenesis and diagnostic criteria for AHR after ABO-i RTx remain unclear. Complement fragment C4d deposition in peritubular capillaries (PTC), which is a sensitive indicator for activation of the classical complement pathway, was studied to establish the pathologic diagnostic indicator of AHR. Methods. Forty-four graft biopsy specimens from 19 patients with ABO-i living donors were analyzed within 90 days after RTx. Nineteen biopsy specimens with acute rejection after ABO-compatible (ABO-c) living-related RTx were used as controls. Diffuse and bright C4d deposition in PTC was considered significantly positive. Results. All of 8 recipients with AHR showed significantly positive C4d in PTC in the ABO-i group, but 9 of 11 recipients without AHR were negative. In the ABO-c RTx group, 16 of 19 recipients were negative for C4d in PTC. The prevalence of C4d in PTC was significantly higher in ABO-i RTx (P <0.05). Conclusions. C4d deposition is valuable as a specific and sensitive indicator for AHR, even of mild severity, in ABO-i RTx.

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.

Enzymatic removal of αGal antigen in pig kidneys by ex vivo and in vivo administration of endo-β-galactosidase C

Xenotransplantation using the pig as a donor species is considered to be a promising solution to the serious shortage of organ donors. Both hyperacute and acute vascular rejection (AVR) are believed to be associated with xenoreactive antibody binding to αGal epitopes on the pig vascular endothelial cells. Thus, suppression of this antigen‐antibody reaction would appear essential for successful long‐term xenograft survival. The purpose of this study was to examine the efficacy of ex vivo and in vivo administration of recombinant endo‐β‐galactosidase C (EndoGalC which, in previous in vitro studies, has been proven to digest αGal antigens completely) on αGal epitopes expressed in pig kidneys. Excised pig kidneys were perfused with University of Wisconsin solution containing EndoGalC and preserved for 4 h. After cold storage, the pig kidney was transplanted into another pig. Ex vivo perfusion and cold storage with EndoGalC reduced αGal epitope expression on vascular endothelial cells to an undetectable level. However, αGal antigens began to be expressed again as early as 1 day after transplantation. The digestion of αGal epitopes by EndoGalC did not cause any damage to the kidney graft. EndoGalC was intravenously administered to two pigs (15 kg), without causing any serious adverse effect. Twelve hours later, >98% of αGal antigens on pig red blood cells (RBCs) had been digested. Immunohistochemical study revealed almost complete elimination of αGal expression on vascular endothelial cells of the kidney graft 4 and 8 h after in vivo administration, but reappearance within 24 h. EndoGalC was administered to a baboon after an interval of 2 months. The second administration did not result in any serious toxicity or reduction in efficacy. These results suggest that ex vivo and in vivo administration of EndoGalC is simple and useful in removing αGal epitopes from pig organs. As the effect of EndoGalC is temporary, multiple in vivo administrations of EndoGalC would be required to inhibit the reappearance of αGal epitopes. Alternatively, transgenic techniques of introducing the gene for EndoGalC into the donor organ might permanently prevent αGal expression.

Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats.

Astrocytes secreting a large amount of 3,4-dihydroxyphenylalanine (dopa) were generated by adenoviral transduction of the human tyrosine hydroxylase (TH) gene. After characterizing in vitro, the effect of transplantation of these astrocytes to the striatum of hemiparkinsonian model rats was investigated. Subconfluent cortical astrocytes were infected by replication-defect adenovirus type 5 carrying the human TH-1 gene or the LacZ reporter gene under the promoter of the glial fibrillary acidic protein (AdexGFAP-HTH-1, AdexGFAP-NL-LacZ). Dopa secretion was not evident at 3 days after the transduction of the HTH-1 gene but it increased from 7 days up to at least 4 months. The secretion was substrate (tyrosine)-dependent, and was enhanced by loading tetrahydrobioputerin (BH4) concentration-dependently. One-third of the hemiparkinsonian model rats, that were transplanted the HTH-1 gene-transduced astrocytes or introduced the direct injection of the viral vector to the striatum, showed a reduction of methamphetamine-induced rotations for at least 6 weeks. Apomorphine-induced rotation was decreased to the 50% level of the controls, but the reduction was obtained equally by the transplantation of HTH-1 gene-transduced or LacZ reporter gene-transduced astrocytes, or by the introduction of HTH-1 or LacZ gene carrying adenovirus. Treatment with FK506 for 3 weeks improved the late-phase apomorphine-induced rotations following the introduction of the HTH-1 gene carrying adenovirus. Histological examination revealed that, in animals that showed a reduction of methamphetamine-rotation, the TH positive astrocytes-like cells were distributed widely in the host striatum for at least 4 weeks. The number of TH positive astrocytes-like cells and their immunoreactivity decreased after 6 weeks when OX-41 positive microglias/macrophages were infiltrated. Data indicate that the adenoviral transduction of the human TH gene to astrocytes and its introduction to the striatum is a promising approach for the treatment of Parkinsons disease. However, the further technical improvements are required to optimize the adenoviral gene delivery, such as the control of viral toxicity and the regulation of the immune response.

Potential value of high‐dose mizoribine as rescue therapy for ongoing acute humoral rejection

Mizoribine (MZ) inhibits the proliferation of lymphocytes selectively via inhibition of inosine monophosphate dehydrogenase, like mycophenolate mofetil (MMF). The clinical dosage of MZ (2–5 mg/kg) is much lower than that of MMF (20–60 mg/kg). The purpose of this study was to examine whether high‐dose MZ would be effective for treatment of acute humoral rejection. Renal transplantation was performed in a different pig strain combination. Group 1 (n = 2) received no treatment. Group 2 (n = 4) received cyclosporine microemulsion (6 mg/kg) and prednisolone (0.1 mg/kg) as baseline immunosuppression. Groups 3 (n = 4), 4 (n = 4) and 5 (n = 4) were additionally treated with MZ for rescue therapy, 30, 10 and 3 mg/kg, respectively, immediately after rejection was observed. All pigs developed acute vascular rejection between days 4 and 8. Complete reversal of acute rejection including reduction of elevated serum creatinine, suppression of anti‐donor antibody production and pathological finding, was obtained in 3/4 (group 3), 1/4 (group 4) and 0/4 (group 5). Rescue with high‐dose MZ (30 mg/kg) reversed ongoing acute humoral rejection. Such a high dose of MZ was tolerable for pigs. However, leukocytopenia was observed when MZ trough level was maintained over 10 μg/ml. Treatment with high‐dose MZ would be applicable to a clinical trial, if blood level is carefully monitored.

Dopamine-denervation enhances the trophic activity in striatum: evaluation by morphological and electrophysiological development in PC12D cells.

To evaluate the possibility that dopamine (DA) denervation enhances the trophic activity in striatum, normal or DA-depleted striatal tissue extract (N- or L-extract, respectively) was obtained, and their trophic effects on PC12D cells were investigated from the viewpoints of differentiation using morphological and electrophysiological analyses. Treatment with N- or L-extract induced neurite outgrowth in a concentration-dependent manner, and induced the enlargement of cell size. These effects were stronger in L-extract than in N-extract. Cation currents were investigated in whole cell patch-clamp mode. Development of cation current started with delayed-rectifier type K+ current (IK) and transient type K+ current (IA), followed by Ca2+ current (ICa) and tetrodotoxin-sensitive Na+ current (INa). INa was expressed more frequently in L-extract treated cells than N-extract treated cells at D7-9. The larger IK amplitude in L-extract treatment at D7-9 seemed to be related to the expression of INa. Development of IA was similar at any stage for both treatments. ICa development started at D3-5 after treatments, and the amplitude and current density were similar in both treatments. ICa was strongly blocked by omega-conotoxin GVIA (3 microM), indicating that N-type channels were mainly expressed after treatments. The data suggests that L-extract has stronger effects to hasten the differentiation of PC12D cells than N-extract by promoting the neurite outgrowth, cell enlargement and expression of voltage-dependent cation channels, especially INa and IK.