Stimulating thoracic epidural placement via a lumbar approach causes significant spinal cord damage in a porcine model.

BACKGROUND: Continuous thoracic epidural analgesia is a valuable and common technique for analgesia but involves risk to the spinal cord. There is significant pediatric experience safely placing thoracic epidurals via a caudal approach. The use of a stimulating catheter offers the advantage of real-time confirmation of appropriate catheter placement. We hypothesize that the tip of a stimulating epidural catheter can be reliably advanced to the thoracic epidural space with lumbar insertion in a porcine model. METHODS: This prospective experimental porcine study evaluated the feasibility of placing the tip of a stimulating epidural catheter to a predefined thoracic epidural location after percutaneous lumbar epidural access in six live pigs. After the lumbar epidural space was accessed, a stimulating epidural catheter was advanced until the targeted thoracic myotome was stimulated. The final position of the catheter in relation to the targeted location was determined by fluoroscopy. All animals were euthanized at the end of the experiment, necropsy and spinal cord histology were then performed to assess the extent of spinal cord damage. RESULTS: In all animals the epidural catheter tip could be accurately advanced to the targeted thoracic myotome. Gross subdural bleeding occurred in three of the six animals and deep spinal damage was observed in two of the six animals. In one animal, the catheter was placed in the subarachnoid space. CONCLUSIONS: Accurate access to the thoracic epidural space is possible via a lumbar approach using a stimulating epidural catheter. Based on gross and histopathological examination, this technique resulted in frequent complications, including subdural hemorrhage, deep spinal cord damage, and subarachnoid catheter placement.

Homogeneous time resolved fluorescence assay to measure histamine release.

Histamine is a biogenic amine synthesized by the enzymatic decarboxylation of histidine. Implication of histamine in allergy is well described but histamine is also found in some specific neurones, functions as a neurotransmitter and regulates sleep/wake cycles, hormonal secretion, cardiovascular control and thermo-regulation. We have developed a TR-FRET histamine assay, based on the competition between sample histamine and allophycocyanine (XL665) labelled histamine for binding to a Europium cryptate (EuK) labelled antibody. As histamine is a small monoamine molecule, high affinity antibodies have been raised against carrier protein conjugated histamine. Therefore, sample histamine needs to be derivatized in the same way as the conjugated histamine, so that the antibody will have a similar affinity for both molecules. This acylation step is performed directly in wells and does not need to be done in separate vials, making handling easier for large numbers of samples. The incubation takes place at room temperature for 3 hours. The assay covers a measurement range of 1.56 to 400 nM and shows an analytical sensitivity of 1.3nM. We have shown that miniaturization of sample and reagents volumes down to 20 micro l does not alter these performances. This histamine release assay provides a particularly well adapted procedure for HTS and secondary screening compared to current heterogeneous methods.

High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous time-resolved fluorescence.

A miniaturized immunoassay for human interleukin-13 (IL-13) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IL-13 which was secreted from NK3.3 cells stimulated with interleukin-2 (IL-2) was detected by measuring the time-resolved fluorescence after adding a mixture of three reagents, biotinylated anti-IL-13 monoclonal antibody, europium cryptate (fluorescence donor)-labeled different anti-IL-13 monoclonal antibody and crosslinked allophycocyanin (fluorescence acceptor)-conjugated with streptavidin in a 384-well assay plate. The detection limit of IL-13 using this immunoassay was estimated to be less than 600 pg/ml and IL-13 levels measured by this method were very close to those measured by enzyme linked immunosorbent assay (ELISA; the correlation coefficient was 0.9535). The proposed assay requires only a fourth of the quantities of all reagents compared with the assay using a conventional 96-well microtiter plate. Furthermore, there is no need to transfer the culture supernatant to another assay plate and wash the plate. Therefore, this miniaturized immunoassay is economical and efficient and is particularly suitable for high-throughput drug screening.