Delia Beju

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

Immunohistochemical and in situ hybridization study of an insulin-like substance in fetal neuron cell cultures

We studied the ability of fetal neuron cell cultures from different rabbit fetal brain gestational ages to produce and secrete an insulin-like substance (ILS). Neurons from 22-day gestation were incubated in serum-containing medium or insulin-free/serum-free medium, and 18-day gestation fetal rabbit neurons were also incubated in serum-free/insulin containing medium and serum-containing medium. The 22-day cultures survived in the serum-containing medium and the insulin-free/serum-free medium. The 18-day cultures died when incubated in the insulin-free/serum-free or serum-free/insulin-containing medium, but survived when incubated in serum-containing medium. Using immunohistochemical and in situ hybridization techniques, ILS and insulin-like mRNA were demonstrated within the 22-day cultures incubated in all media compositions, but not within the 18-day cultures incubated in the serum-containing medium. Ultrastructural studies of the 22-day cultures demonstrated an ILS in the endoplasmic reticulum, Golgi and cytoplasm. Northern blots showed the presence of an insulin-like mRNA within the 22-day gestation neuron cell cultures. Insulin receptor was present in the 22-day cultures, but was absent in the 18-day cultures. In addition, we characterized the ILS from the 22-day cultures incubated in the insulin-free/serum-free medium employing high-performance liquid chromatography (HPLC), radioimmunoassay and Western blots. Analysis by HPLC and Western blots demonstrated the presence of an ILS in the extract. We have shown that while 22-day fetal neuron cultures produce and secrete an insulin-like substance indistinguishable from authentic insulin, neuron cell cultures from early brain development do not express this capability.

Clearance of technetium 99m N-NOET in normal, ischemic-reperfused, and membrane-disrupted myocardium

BackgroundTechnetium 99m-labeled bis (N-ethoxy, N-ethyl dithiocarbamato) nitrido technetium(v) (99mTcN-NOET) is a new neutral cardiac perfusion imaging agent that has been shown to have very high uptake and retention in vitro. The purpose of this study was to determine the clearance kinetics of 99m TcN-NOET in control, ischemic-reperfused, and membrane-disrupted myocardium.Methods and ResultsAfter a 100 μCi (3.7×106 Bq) bolus of 99mTcN-NOET was injected, myocardial clearance was monitored for 1 hour by the use of a sodium iodide detector in 30 isolated, Krebs-Henseleit (KH) perfused rat hearts. Seven hearts were used as controls (group 1). In seven ischemic-reperfused hearts, tracer administration and uptake was followed by 30 minutes of no flow and 1 hour of reflow (group 2). In six additional ischemic-reperfused hearts, tracer administration was followed by deprivation of flow for 1 hour followed by 1 hour of reflow (group 3). Six hearts were perfused with a 0.5% Triton X-100 KH perfusate for 1 hour (group 4). Four hearts were perfused with KH for 10 minutes, followed by cyanide for 10 minutes (group 5). This cycle was repeated three times. Activities remaining in each heart at the end of each experiment were quantitated, and activity at peak uptake was calculated. The 99mTcN-NOET myocardial clearance was near linear in the control (0.6±0.4) and both ischemic-reperfused groups with virtually no fractional clearance (1.2%±0.6% and 2.1%±0.6%, respectively; p=NS). In the Triton X-100 membrane-disrupted hearts, clearance was substantial (94.2%±4.0%; p<0.0001 compared with the control and ischemic-reperfused groups). Cyanide treatment produced rapid clearance, which was arrested by a return to the standard KH perfusate. Peak uptake as a percentage of injected dose was 74.9%±1.4% for all groups combined.ConclusionThus 99mTcN-NOET has extremely high myocardial retention after 1 hour in normal myocardium and is not significantly affected by ongoing myocardial ischemia or reperfusion injury in this model. Clearance is increased markedly in extreme conditions of membrane disruption. These data are consistent with the concept that 99mTc-NOET is localized predominantly in or on cell membranes. 99mTcN-NOET is a promising, new myocardial perfusion imaging agent that exhibits a stable myocardial distribution in the setting of acute developing injury.

HL-91-Technetium-99m: A new marker of viability in ischemic myocardium

BackgroundTechnetium 99m-HL91 is a new hypoxia imaging agent that demonstrates increased uptake in ischemic, viable myocardium. This study was performed to determine whether HL91 is taken up by nonviable myocardium.MethodsTwenty-three Krebs-Henseleit buffer-perfused, isolated rat hearts were studied. Tc-99m-HL91 300 μCi was infused over 10 minutes, followed by a 60-minute clearance. Myocardial activity was monitored by use of an NaI crystal. Four groups were studied: control (flow=12 mL/min, n=7), low flow (flow=1 mL/min, n=6), no flow/reflow (60 minutes no flow/60 minutes reflow before Tc-99m-HL91 infusion, flow=12 mL/min, n=5), and cyanidetreated (before Tc-99m-HL91 infusion, flow=12 mL/min, n=5). Injury was assessed by creatine kinase, transmission electron microscopy, and triphenyltetrazolium chloride.ResultsControl (no injury) and cyanide-treated (severe injury) hearts demonstrated low uptake (6.3±0.5 mean±SEM and 5.7±1.2 μCi, respectively) and low 60-minute retention (13.8%±2.2% and 13.7%±3.9%, respectively). Low-flow hearts (minimal injury) demonstrated markedly increased uptake (43.5±2.8 μCi, P<.01) and increased 60-minute retention (33.2% ±2.9%, P<.01) compared with control. No-flow/reflow hearts (moderate injury) demonstrated intermediate uptake (8.7±0.5 μCi, P<.05 to control), although retention was not significantly different (18.9%±3.5%, P=ns). Severely and rapidly injured myocardium demonstrated Tc-99m-HL91 peak uptake and retention indistinguishable from normal. Moderately injured myocardium demonstrated uptake intermediate between severely injured and low-flow-induced ischemic, viable myocardium.ConclusionThus Tc-99m-HL91 is not taken up or retained in nonviable and irreversibly injured myocardium.

An immunohistochemical and in situ hybridization study of insulin-like growth factor I within fetal neuron cell cultures

Fetal neuron cell cultures (NCC) from 22 day gestation and 18 day gestation fetal rabbit brain were studied for the presence of insulin-like growth factor I (IGF I). The 22 day gestation NCC were incubated in an IGF I free/insulin free/serum free medium. The 18 day gestation NCC were incubated in: (1) IGF I free/insulin free/serum free medium, (2) IGF I containing medium (100 ng)/serum free medium, and (3) serum containing medium. The 22 day gestation NCC survived in the IGF I free/insulin free/serum free medium. Furthermore, IGF I was detected in the medium by RIA from day one to day ten of incubation. In contrast, the 18 day gestation NCC did not survive in the IGF I free/insulin free/serum medium, but survived in the serum medium. When the 18 day gestation NCC were incubated in the serum free medium containing 100 ng IGF I the cells survived for a period of 2-3 days. Immunoreactive IGF I was found within the 22 day gestation NCC incubated in the IGF I free/insulin free/serum free medium and 18 day gestation NCC in serum medium. Likewise, IGF I mRNA was found only within the 22 day gestation NCC. Internalization studies of IGF I have shown that the peptide was internalized from the medium by the two different gestational age NCCs studied. IGF I receptors were found in both 22 day gestation and 18 day gestation NCC. In conclusion IGF I may promote cell survival in early stages of brain development, and may be of exogenous origin. In contrast the 22 day gestation NCC are capable of producing and secreting IGF I, and indeed appear to respond to this growth factor in an autocrine fashion.