Visualization of lower extremity lymphedema in the same cohort using 99mTc-human serum albumin and 99mTc-phytate lymphoscintigraphy with SPECT-CT.

Lymphoscintigraphy with single-photon emission computed tomography (SPECT-CT) is useful in diagnosing lymphedema. However, there are multiple timings, techniques, and tracers utilized worldwide without any comparison. We examined and compared the image clarity with two different radiotracers, 99mTc human serum albumin (HSA) and 99mTc phytate (phytate), in the same patients. The study retrospectivity examined 46 limbs of 36 patients who underwent lymphoscintigraphy using HSA and phytate from January 2013 to September 2018. Tracer accumulation in the lymph nodes, linear pattern (LP), and dermal backflow (DBF) were qualitatively analyzed; contrast-to-noise ratios (CNR) of DBF and standardized uptake value ratio (SUVR) of LP were also quantitatively analyzed. Neither lymph node accumulation nor DBF identification showed significant difference. However, a significant difference was observed between the LP identification of the unaffected (p<0.001) and affected sides (p<0.001). On quantitative evaluation, CNR and SUVR of LP was significantly higher with HSA than with phytate (p<0.001). SUVR of LP was also significantly higher with HSA than with phytate in both unaffected (p=0.002) and affected (p=0.005) sides. Overall, images acquired with HSA were clearer than that with phytate, and the identification of LP was particularly better with HSA than with phytate. Thus, lymphoscintigraphy using HSA is preferred over phytate for both diagnosis and evaluation of disease severity and surgical site selection.

Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer's disease therapy.

Here, we report a novel target of the drug memantine, ATP-sensitive K+ (KATP) channels, potentially relevant to memory improvement. We confirmed that memantine antagonizes memory impairment in Alzheimer's model APP23 mice. Memantine increased CaMKII activity in the APP23 mouse hippocampus, and memantine-induced enhancement of hippocampal long-term potentiation (LTP) and CaMKII activity was totally abolished by treatment with pinacidil, a specific opener of KATP channels. Memantine also inhibited Kir6.1 and Kir6.2 KATP channels and elevated intracellular Ca2+ concentrations in neuro2A cells overexpressing Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed at postsynaptic regions of hippocampal neurons, whereas Kir6.1 was predominant in dendrites and cell bodies of pyramidal neurons. Finally, we confirmed that Kir6.2 mutant mice exhibit severe memory deficits and impaired hippocampal LTP, impairments that cannot be rescued by memantine administration. Altogether, our studies show that memantine modulates Kir6.2 activity, and that the Kir6.2 channel is a novel target for therapeutics to improve memory impairment in Alzheimer disease patients.

Endocytosis following dopamine D2 receptor activation is critical for neuronal activity and dendritic spine formation via Rabex-5/PDGFRß signaling in striatopallidal medium spiny neurons.

Aberrant dopamine D2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/platelet-derived growth factor receptor-ß (PDGFRß)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFRß are then transported to the Golgi apparatus, where those complexes trigger Gαi3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFRß is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.

Nobiletin treatment improves motor and cognitive deficits seen in MPTP-induced Parkinson model mice.

Nobiletin, a polymethoxylated flavonoid found in citrus fruit peel, reportedly improves memory impairment in rodent models. Here we report its effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced motor and cognitive deficits. Nobiletin administration (50mg/kg i.p.) for 2 consecutive weeks improved motor deficits seen in MPTP-induced Parkinson model mice by 2weeks, an effect that continued until 2weeks after drug withdrawal. Drug treatment promoted similar rescue of MPTP-induced cognitive impairment at equivalent time points. Nonetheless, nobiletin treatment did not block loss of dopaminergic neurons seen in the MPTP-treated mouse midbrain, nor did it rescue decreased tyrosine hydroxylase (TH) protein levels seen in the striatum or hippocampal CA1 region of these mice. Interestingly, nobiletin administration (50mg/kg i.p.) rescued reduced levels of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and phosphorylation at Thr-34 of dopamine- and cAMP-regulated phosphoprotein-32 (DARPP-32) in striatum and hippocampal CA1 to levels seen in sham-operated mice. Likewise, CaMKII- and cAMP kinase-dependent TH phosphorylation was significantly restored by nobiletin treatment. MPTP-induced reduction of dopamine contents in the striatum and hippocampal CA1 region was improved by nobiletin administration (50mg/kg i.p.). Acute intraperitoneal administration of nobiletin also enhanced dopamine release in striatum and hippocampal CA1, an effect partially inhibited by treatment with nifedipine (a L-type Ca(2+) channel inhibitor) or NNC 55-0396 (a T-type Ca(2+) channel inhibitor) and completely abolished by combined treatment with both. Overall, our study describes a novel nobiletin activity in brain and suggests that nobiletin rescues motor and cognitive dysfunction in MPTP-induced Parkinson model mice, in part by enhancing dopamine release.

Oral administration of glutathione improves memory deficits following transient brain ischemia by reducing brain oxidative stress.

Oxidative stress aggravates brain injury following ischemia. The glutathione (GSH) system plays a pivotal role in combating oxidative stress in various cell types. To determine whether oral GSH administration elicits anti-oxidative effects, we assessed its potential neuroprotective effects in transient bilateral common carotid artery occlusion (BCCAO) mice. In naïve mice, acute oral administration of GSH significantly increased GSH levels by 1h in the cortex and hippocampus. Eleven days after BCCAO, untreated mice showed significantly decreased GSH levels and an inverse elevation of glutathione-disulfide (GSSG) levels in both the cortex and hippocampus. Oral administration of GSH (100 and 500 mg/kg p.o.) for 10 consecutive days after ischemia restored reduced GSH levels and inhibited GSSG elevation. Notably, post-administration of GSH (100 and 500 mg/kg p.o.) significantly prevented neuronal cell death in the hippocampal CA1 region in BCCAO mice, an effect closely correlated with decreased levels of oxidative markers such as 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2-deoxyguanosine (8-OHdG) and nitrotyrosine in that region. Finally, GSH administration for 10 days improved memory deficits observed in BCCAO mice. Taken together, our findings indicate that the anti-oxidative effect of oral GSH administration ameliorates post-ischemia neuronal cell death and, in turn, may improve memory.

Decreased CaMKII and PKC activities in specific brain regions are associated with cognitive impairment in neonatal ventral hippocampus-lesioned rats.

Neonatal ventral hippocampus (NVH)-lesioned rats represent a neurodevelopmental impairment model of schizophrenia. Previous observations indicate that postpubertal NVH-lesioned rats exhibit impairments in prepulse inhibition (PPI), spontaneous locomotion and social interaction behavior. Here, we document the neurochemical basis of those defects. PPI impairment but not cognitive impairment was improved by acute risperidone treatment (0.30mg/kgi.p.). Immunohistochemical analyses using anti-autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) antibody indicated significantly reduced CaMKII autophosphorylation, especially in the medial prefrontal cortex (mPFC), striatum and hippocampal CA1 region, of NVH-lesioned rats relative to control animals. We also confirmed that reduced CaMKII autophoshorylation in the mPFC, striatum and hippocampal CA1 region causes decreased phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolpropionic acid-type glutamate receptor subunit 1 (GluR1) (Ser 831), a CaMKII substrate. Like CaMKII, PKCα (Ser 657) autophosphorylation and NR1 (Ser 896) phosphorylation were decreased both in the mPFC and CA1 region. Interestingly, phosphorylation of DARPP-32 (Thr 34) was decreased in the mPFC but increased in the striatum and CA1 region of NVH-lesioned rats compared to controls. Risperidone treatment restored increased DARPP-32 phosphorylation in the striatum and CA1 regions of NVH-lesioned rats but did not rescue CaMKII and PKCα autophosphorylation. Taken together, we find that impaired cognition observed in NVH-lesioned rats is associated with decreased CaMKII and PKCα activities in memory-related brain regions, changes not rescued by risperidone treatment.

Human elastic cartilage engineering from cartilage progenitor cells using rotating wall vessel bioreactor.

Transplantation of bioengineered elastic cartilage is considered to be a promising approach for patients with craniofacial defects. We have previously shown that human ear perichondrium harbors a population of cartilage progenitor cells (CPCs). The aim of this study was to examine the use of a rotating wall vessel (RWV) bioreactor for CPCs to engineer 3-D elastic cartilage in vitro. Human CPCs isolated from ear perichondrium were expanded and differentiated into chondrocytes under 2-D culture conditions. Fully differentiated CPCs were seeded into recently developed pC-HAp/ChS (porous material consisted of collagen, hydroxyapatite, and chondroitinsulfate) scaffolds and 3-D cultivated utilizing a RWV bioreactor. 3-D engineered constructs appeared shiny with a yellowish, cartilage-like morphology. The shape of the molded scaffold was maintained after RWV cultivation. Hematoxylin and eosin staining showed engraftment of CPCs inside pC-HAp/ChS. Alcian blue and Elastica Van Gieson staining showed of proteoglycan and elastic fibers, which are unique extracellular matrices of elastic cartilage. Thus, human CPCs formed elastic cartilage-like tissue after 3-D cultivation in a RWV bioreactor. These techniques may assist future efforts to reconstruct complicate structures composed of elastic cartilage in vitro.

Classification of lymphoscintigraphy and relevance to surgical indication for lymphaticovenous anastomosis in upper limb lymphedema.

Upper limb lymphedema that develops after breast cancer surgery causes physical discomfort and psychological distress, and it can require both conservative and surgical treatment. Lymphaticovenous anastomosis has been reported to be an effective treatment; however the disease severity criteria that define indications for this treatment remain unclear. Here, we examined lymphoscintigraphic findings in 78 patients with secondary upper limb lymphedema and classified them into 5 major types (Type I-V) and 3 subtypes (Subtype E, L, and 0). Results revealed that this classification is related to the clinical stage scale of the International Society of Lymphology. Based on intraoperative examination findings in 20 of the 78 patients, lymphatic pressure is likely to be further elevated in Type II-V cases which are characterized by the presence of dermal back flow. Therefore, lymphaticovenous anastomosis should be considered as a treatment option for lymphedema in Type II-V cases. Furthermore, there are only limited lymph vessel sites usable for lymphaticovenous anastomosis in more severe lymphedema types [Types IV and Type V (which is characterized by dermal backflow only in the hand)]. The findings in Type IV-V cases suggest that therapeutic strategies for severe upper limb lymphedema need further consideration.

The RING heterodimer BRCA1-BARD1 is a ubiquitin ligase inactivated by a breast cancer-derived mutation.

BRCA1-BARD1 constitutes a heterodimeric RING finger complex associated through its N-terminal regions. Here we demonstrate that the BRCA1-BARD1 heterodimeric RING finger complex contains significant ubiquitin ligase activity that can be disrupted by a breast cancer-derived RING finger mutation in BRCA1. Whereas individually BRCA1 and BARD1 have very low ubiquitin ligase activities in vitro, BRCA1 combined with BARD1 exhibits dramatically higher activity. Bacterially purified RING finger domains comprising residues 1-304 of BRCA1 and residues 25-189 of BARD1 are capable of polymerizing ubiquitin. The steady-state level of transfected BRCA1 in vivo was increased by co-transfection of BARD1, and reciprocally that of transfected BARD1 was increased by BRCA1 in a dose-dependent manner. The breast cancer-derived BARD1-interaction-deficient mutant, BRCA1(C61G), does not exhibit ubiquitin ligase activity in vitro. These results suggest that the BRCA1-BARD1 complex contains a ubiquitin ligase activity that is important in prevention of breast and ovarian cancer development.

Radical hysterectomy: An anatomic evaluation of parametrial dissection.

OBJECTIVES: This study was designed to demonstrate a reduction in the amount of blood loss for vesicouterine ligament dissection and to investigate the intrapelvic autonomic nerve pathway and its preservation by means of anatomic analysis. METHODS: The anchoring mechanism of the pelvic viscera to the pelvic wall was divided into a supporting system facing laterally and a suspensory system facing dorsoventrally. An operative procedure was designed in which both systems were separated and dissected independently. RESULTS: Between the two systems, an artificial space was developed, which required a new dissection method for the parametrium and revealed a new anatomic pathway for the ureter and autonomic nerve. The amount of blood loss (mean +/- SD) during dissection of the vesicouterine ligament was ultimately 260.1 +/- 114.8 ml. Postoperatively, the maximum capacity of the bladder was 393.9 +/- 40.4 ml, maximum detrusor pressure 6.3 +/- 4.1 cm H(2)O, mean compliance >10 ml/cm H(2)O, residual urine 23.8 +/- 9.4 ml, and maximum flow rate 25. +/- 8 2.2 ml/s, respectively. CONCLUSION: A new classification for the parametrium and its dissection method have been established. Development of this new operative procedure has also contributed to a decrease in blood loss and preservation of bladder function.

Myofibroblastoma of the neck in a heifer.

A 1.5-year-old Holstein heifer had a subcutaneous tumor mass (20 cm diameter) on the ventral portion of the neck, and the tumor was diagnosed as a locally invasive myofibroblastoma. It consisted of moderately cellular fibrous tissue, and the interlobular septum of the thymus was invaded by tumor cells. The neoplastic cells were positive for alpha smooth muscle actin and vimentin, but not for desmin. Electron microscopy disclosed the presence of moderately developed rough endoplasmic reticulum and microfilaments with focal densities.

[Decreasing seropositivity of cytomegalovirus of pregnant women in Japan].

We examined seroprevalence of cytomegalovirus (CMV) of pregnant women for 18 years since 1980 with complement-fixing antibody (CF) and specific IgG antibody. CF seropositive rate decreased gradually from 93.2% to 66.7%. CMV-IgG Seropositive rates were 87.4% in 1985, and 75.2% in 1996 to 1997. Age and parity of pregnant women associated with the immune status; 35.6% of recent young primipara were susceptible.

A new proposal for radical hysterectomy.

OBJECTIVE: The authors revised the surgical procedure for radical hysterectomy utilizing detailed observation of the venous system, connective fascial sheets, and neural pathways within the uterine supports. STUDY DESIGN: The anterior, middle, and posterior uterine supports were reclassified into two systems, supporting or fascial and drainage or areoral. Supporting system consisted of the superficial layer of the vesicouterine ligament, fascial part of the Mackenrodt ligament, sacrouterine ligament, and rectovaginal ligament, whereas drainage system consisted of the deep layer of the vesicouterine ligament, vascular part of the Mackenrodt ligament, and the so-called mesoureter. The operative procedure was planned according to the continuity of these ligaments and executed first by excising the fascia and then dissecting the denuded areoral tissue. RESULTS: Among the 15 patients who underwent surgery for uterine cancer during a 2-year period the mean (SD) time required for the operation was 305.5 +/- 30.5 min and the mean (SD) total volume of blood loss 592.0 +/- 238.2 ml. A mean (SD) period of 14.3 +/- 3.8 postoperative days was required until the volume of the residual urine decreased to less than 50 mL. CONCLUSION: The present operation has been structured more three-dimensionally and systematically than before. Further, safety of the operation was significantly improved including prevention of hemorrhage and preservation of bladder function.

[Simplified avidity assay of rubella IgG antibody in rubella virus infection].

Simplified avidity assay of rubella IgG antibody with urea was evaluated to distinguish primary rubella from reinfection. In this method urea washing was done once for 10 minutes. The avidity index (AI) was calculated as the optical density percentage for the urea-washed well when compared to that of the non-treated well. We examined 292 sera from 50 patients with primary infection collected 6 to 2,259 days after the rash appeared, 29 sera from 11 patients with rubella reinfection and 69 sera from 68 pregnant women without fetal infection and having a high hemagglutination inhibition (HI) antibody. In primary infection AI increased gradually from 0%, and reached a plateau of about 60% four months after the rash appeared, whereas the mean AIs of patients after reinfection and with high HI antibody were as high as 87.1% and 89.9%, respectively. These results indicate that the simplified avidity assay in rubella IgG antibody is also valuable in diagnosing recent primary rubella in pregnant women with a high HI antibody.

[Avidity of rubella IgG antibody in rubella virus infection].

The avidity assay of rubella IgG antibody with urea was evaluated to distinguish the early convalescent phase of primary rubella from the late one and reinfection. We examined 116 sera from 23 patients with primary infection collected 7 to 1,477 days after the rash appeared and 25 sera from 8 patients with rubella reinfection and 16 sera from 15 pregnant women without fetal infection having high hemagglutination inhibition (HI) antibody or weak positive rubella IgM antibody. The avidity index (AI) was calculated as the ratio of the optical density from the urea-washed well to that of the non-treated well, expressed as a percentage. In primary infection AI were lower than 30% within 30 days after the rash appeared, then gradually increased and reached a plateau of 60 to 80% three months later, whereas the AIs of patients after reinfection, with high HI antibody and weak positive IgM antibody were as high as 79.2 +/- 7.5%, 93.5 +/- 4.1% and 90.6 +/- 5.0%, respectively. These results indicate that the measurement of avidity in rubella IgG antibody is valuable in diagnosing recent primary rubella in pregnant women having a high HI antibody or IgM antibody.

Dissection of the cardinal ligament in radical hysterectomy for cervical cancer with emphasis on the lateral ligament.

Surgical experience with carcinomas of the uterus and rectum has provided new insights into the surgical anatomy of a lamina, which separates the paravesical space from the pararectal space. It has been proved that each of the lamina consists of the cardinal and lateral ligaments and pelvic splanchnic nerves, descending in the following order. The cardinal and lateral ligaments, as a connective stalk, insert into the lateral walls of the uterus and rectum extending from the inner aspect of the pelvic wall. Clarification of this structural relationship led to the development of a new procedure for the dissection of the cardinal ligament in radical hysterectomy, while still preserving the lateral ligament. This facilitated systematic dissection of the cardinal and uterosacral ligaments with posterior manipulation, leading to a reduction in blood loss and to prevention of brisk bleeding from the venous plexuses.

Purification of a kininogenase (kininogenase-2) from the venom of Agkistrodon caliginosus (Kankoku-Mamushi).

From the partially purified capillary permeability-increasing enzyme obtained from A. caliginosus venom, another kininogenase (kininogenase-2) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50, S-Sepharose Fast Flow and Q-Sepharose Fast Flow. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and SDS-gel electrophoresis. The kininogenase-2 had arginine ester hydrolytic and capillary permeability-increasing activities, and did not show any caseinolytic or clotting activity in a similar manner to a previously purified kininogenase (kininogenase-1). The purified kininogenase-2 liberated bradykinin on incubation with purified bovine high mol. wt kininogen. The rate of bradykinin release from the kininogen by kininogenase-2 was slower than that by the kininogenase-1, although both enzymes rapidly cleaved the peptide bonds in the kininogen molecule.