[Surgical technique and outcomes of uteri retrieval from brain-dead multi-organ donors: a preclinical research of human uterine transplantation].

Objective: To evaluate the surgical technique and outcomes of uteri retrieval from brain-dead multi-organ donors.This study is a preclinical research of human living uterine transplantation. Methods: From May, 2015 to May, 2017, four uteri retrieval procedures, characterized with radical hysterectomy and uterine vascular pedicles dissection, were performed in multi-organ brain-dead donors.The uterus was the third authorized organ after the kidney and liver retrieval procedures in the first two cases.The uterine pedicles included the uterus, ovaries, fallopian tubes, the upper one-third of the vagina and internal iliac vessels or external iliac vessels.The perfusion of the uterus was conducted after the retrieval for evaluating the availability, followed by histopathological examination of the uterine issues per 30 minutes. Results: Since the uterine vein was quite difficult to identify and dissect in the first two case, which result in the rupture of triple uterine veins.Therefore, the uterine venous vessels including uterine vein connected with internal iliac vein and internal iliac arteries were selected as vascular grafts and dissected successfully in the last two cases, which could be perfused with the mixture of 4 ℃ heparinized physiological saline through each artery because of shortening the surgical time and arranging the uterine procurement as the first authorized organ procedure.Mean (SD) operative time was 152.5±39.0 min (115-215 min, n=4). Conclusion: Our preliminary experience indicated that the uterus could be retrieved from the brain-dead multi-organ donors and transplanted to the recipient.The attempt of skeletonizing the uterine veins should be replaced by dissection of internal iliac vein.

Impaired Albumin Uptake and Processing Promote Albuminuria in OVE26 Diabetic Mice.

The importance of proximal tubules dysfunction to diabetic albuminuria is uncertain. OVE26 mice have the most severe albuminuria of all diabetic mouse models but it is not known if impaired tubule uptake and processing are contributing factors. In the current study fluorescent albumin was used to follow the fate of albumin in OVE26 and normal mice. Compared to normal urine, OVE26 urine contained at least 23 times more intact fluorescent albumin but only 3-fold more 70 kD fluorescent dextran. This indicated that a function other than size selective glomerular sieving contributed to OVE26 albuminuria. Imaging of albumin was similar in normal and diabetic tubules for 3 hrs after injection. However 3 days after injection a subset of OVE26 tubules retained strong albumin fluorescence, which was never observed in normal mice. OVE26 tubules with prolonged retention of injected albumin lost the capacity to take up albumin and there was a significant correlation between tubules unable to eliminate fluorescent albumin and total albuminuria. TUNEL staining revealed a 76-fold increase in cell death in OVE26 tubules that retained fluorescent albumin. These results indicate that failure to process and dispose of internalized albumin leads to impaired albumin uptake, increased albuminuria, and tubule cell apoptosis.

Mosaic SCN1A mutations in familial partial epilepsy with antecedent febrile seizures.

SCN1A is the most relevant epilepsy gene. Mutations of SCN1A generate phenotypes ranging from the extremely severe form of Dravet syndrome (DS) to a mild form of generalized epilepsy with febrile seizures plus (GEFS+). Mosaic SCN1A mutations have been identified in rare familial DS. It is suspected that mosaic mutations of SCN1A may cause other types of familial epilepsies with febrile seizures (FS), which are more common clinically. Thus, we screened SCN1A mutations in 13 families with partial epilepsy with antecedent febrile seizures (PEFS+) using denaturing high-performance liquid chromatography and sequencing. The level of mosaicism was further quantified by pyrosequencing. Two missense SCN1A mutations with mosaic origin were identified in two unrelated families, accounting for 15.4% (2/13) of the PEFS+ families tested. One of the mosaic carriers with ~25.0% mutation of c.5768A>G/p.Q1923R had experienced simple FS; another with ~12.5% mutation of c.4847T>C/p.I1616T was asymptomatic. Their heterozygous children had PEFS+. Recurrent transmission occurred in both families, as noted in most of the families with germline mosaicism reported previously. The two mosaic mutations identified in this study are less destructive missense, compared with the more destructive truncating and splice-site mutations identified in the majority of previous studies. This is the first report of mosaic SCN1A mutations in families with probands that do not exhibit DS, but manifest only a milder phenotype. Therefore, such families with mild cases should be approached with caution in genetic counseling and the possibility of mosaicism origin associated with high recurrence risk should be excluded.

Altered expression of voltage-gated potassium channel 4.2 and voltage-gated potassium channel 4-interacting protein, and changes in intracellular calcium levels following lithium-pilocarpine-induced status epilepticus.

The A-type voltage-gated potassium channels (Kv4) have been proved to play a major role as modulators of somatodendritic excitability. Recent studies indicate that neuronal hyperactivity in epilepsy is associated with changes in Kv4. However, the precise regulation of Kv4 in the development of epilepsy and its underlying mechanism remain unclear. In this study, we investigated whether the expression of the Kv4.2 channel and of its major modulator, voltage-dependent potassium channel-interacting protein (KChIP1), is altered following lithium-pilocarpine induced status epilepticus (SE) and the chronic-epilepsy phase in the rat model. We found that Kv4.2 and KChIP1 expression was transiently up-regulated following SE, whereas it was down-regulated during the chronic phase: this was most prominent in the CA1 and CA3 regions. The time-course analysis of the protein expression level showed that the peak Kv4.2 up-regulation was between 6 and 24 h after SE, whereas KChIP1 expression was increased earlier and for a shorter period. The temporospatial changes in Kv4.2 were very similar to those of its major modulator KChIP1. We compared the difference in 4-aminopyridine (4-AP)-induced intracellular calcium ([Ca(2+)]i) elevation between model and control brain slices. The results showed that the [Ca(2+)]i elevation induced by the Kv4 channel blocker 4-AP was aggravated and prolonged in the model slice after SE. The functional relevance of these changes in Ca(2+) homeostasis and Kv4.2 and KChIP1 expression may be associated with intrinsic neuronal excitability regulation and epileptogenesis.

The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells.

AIMS/HYPOTHESIS: Pyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure. METHODS: To confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells. RESULTS: Treatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP(+) ratio and activity of the pyruvate-malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion. CONCLUSIONS/INTERPRETATION: Our findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation.

Malic enzyme is present in mouse islets and modulates insulin secretion.

AIMS/HYPOTHESIS: The pyruvate-malate shuttle is a metabolic cycle in pancreatic beta cells and is important for beta cell function. Cytosolic malic enzyme (ME) carries out an essential step in the shuttle by converting malate to pyruvate and generating NADPH. In rat islets the pyruvate-malate shuttle may regulate insulin secretion and it has been shown to play a critical role in adaptation to obesity and insulin resistance. However, ME has not been demonstrated in mouse islets and three reports indicate that mouse islets contain no ME activity. If mouse islets lack ME, rat and mouse islets must regulate insulin secretion by different mechanisms. METHODS: We measured ME activity by a fluorometric enzymatic assay and Me mRNA by real-time PCR. ME activity was also measured in streptozotocin-treated mouse islets. FACS-purified beta cells were obtained from MIP-GFP mouse islets, agouti-L obese mouse islets and mouse beta cell line MIN-6. Insulin secretion and NADPH/NADP(+) ratios were measured in Me siRNA-treated beta cells. RESULTS: ME activity and Me mRNA were present in C57BL/6 mouse islets. ME activity was reduced in streptozotocin-treated mouse islets. ME activity was also measurable in FACS-purified mouse beta cells. In addition, ME activity was significantly increased in obese agouti-L mouse islets and the mouse MIN-6 cell line. Me siRNA inhibited ME activity and reduced glucose-stimulated insulin secretion and also inhibited NADPH products. CONCLUSIONS/INTERPRETATION: Mouse islets contain ME, which plays a significant role in regulating insulin secretion.

Enhanced rat beta-cell proliferation in 60% pancreatectomized islets by increased glucose metabolic flux through pyruvate carboxylase pathway.

Islet beta-cell proliferation is a very important component of beta-cell adaptation to insulin resistance and prevention of type 2 diabetes mellitus. However, we know little about the mechanisms of beta-cell proliferation. We now investigate the relationship between pyruvate carboxylase (PC) pathway activity and islet cell proliferation 5 days after 60% pancreatectomy (Px). Islet cell number, protein, and DNA content, indicators of beta-cell proliferation, were increased two- to threefold 5 days after Px. PC and pyruvate dehydrogenase (PDH) activities increased only approximately 1.3-fold; however, islet pyruvate content and malate release from isolated islet mitochondria were approximately threefold increased in Px islets. The latter is an indicator of pyruvate-malate cycle activity, indicating that most of the increased pyruvate was converted to oxaloacetate (OAA) through the PC pathway. The contents of OAA and malate, intermediates of the pyruvate-malate cycle, were also increased threefold. PDH and citrate content were only slightly increased. Importantly, the changes in cell proliferation parameters, glucose utilization, and oxidation and malate release were partially blocked by in vivo treatment with the PC inhibitor phenylacetic acid. Our results suggest that enhanced PC pathway in Px islets may have an important role in islet cell proliferation.