Establishing a through-puncture model for assessing post-injection leakage in the intervertebral disc.

PURPOSE: Needle injection through the outer annulus fibrosus of the intervertebral disc (IVD) is the most practical approach for delivery of therapeutic agents, which have been shown to potentially leak following needle retraction. The goal of this work was to establish a protocol for quantifying post-injection leakage and test its sensitivity to factors believed to affect needle track geometry. METHODS: A through-puncture defect procedure, followed by controlled injection, was performed on bovine caudal IVDs. Sensitivity to needle size was tested by injection of saline into unconstrained discs with either a 30G, 26G, or 21G hypodermic needle. Sensitivity to axial load was tested by repeated injection via a 26G needle with either no constraint, fixed height, or 10% axial compressive strain. Sensitivity to flexion was tested by applying combined 0.2 MPa compression and 15° of flexion following injection of 5% of disc volume. RESULTS: Needle diameter significantly affected maximum volume prior to leakage, ranging from 34.6 ± 31.9 µL when using 21G to 115.6 ± 23.6 µL when using 30G. While all unloaded discs leaked, axial compression decreased the incidence of leakage events by 50-100% depending on load history. Forward flexion resulted in a 22% incidence of leakage. CONCLUSION: Fluid injected into IVDs is at significant risk of leakage following needle retraction. This risk depends on factors which alter the geometry of the needle track, including needle size, pinching due to axial compression, and stretching as a result of forward flexion.

Simultaneous detection of multiple mRNAs and proteins in bovine IVD cells and tissue with single cell resolution.

OBJECTIVES: Interactions of cells with their neighbors and influences by the surrounding extracellular matrix (ECM) is reflected in a cells transcriptome and proteome. In tissues comprised of heterogeneous cell populations or cells depending on ECM signalling cues such as those of the intervertebral disc (IVD), this information is obscured or lost when cells are pooled for the commonly used transcript analysis by quantitative PCR or RNA sequencing. Instead, these cells require means to analyse RNA transcript and protein distribution at a single cell or subcellular level to identify different cell types and functions, without removing them from their surrounding signalling cues. RESULTS: We developed a simple, sequential protocol combining RNA is situ hybridisation (RISH) and immunohistochemistry (IHC) for the simultaneous analysis of multiple transcripts alongside proteins. This allows one to characterize heterogeneous cell populations at the single cell level in the natural cell environment and signalling context, both in vivo and in vitro. This protocol is demonstrated on cells of the bovine IVD, for transcripts and proteins involved in mechanotransduction, stemness and cell proliferation. CONCLUSIONS: A simple, sequential protocol combining RISH and IHC is presented that allows for simultaneous information on RNA transcripts and proteins to characterize cells within a heterogeneous cell population and complex signalling environments such as those of the IVD.

Slow depressurization following intradiscal injection leads to injectate leakage in a large animal model.

Needle injection has been indicated as the most practical method of delivering therapeutic agents to the intervertebral disc due to the disc's largely avascular nature. As the disc is characterized by both high stiffness and low permeability, injection requires substantial pressure, which may not relax on practical time scales. Additionally, needle puncture results in a localized disruption to the annulus fibrosus that can provide a leakage pathway for pressurized injectate. We hypothesized that intradiscal injection would result in slow relaxation of injectate pressure, followed by leakage upon needle retraction. This hypothesis was tested via controlled injection of fluorescently labeled saline into bovine caudal discs via a 21 gauge needle. Injections were performed with 10% of total disc volume injected at 3%/s followed by a 4-minute dwell. An analytical poroelastic model was calibrated to the experimental data and used to estimate injectate delivery with time. Experimental results confirmed both pressurization (with a peak of 199 ± 45 kPa) and slow recovery (final pressure of 81 ± 23 kPa). Injectate leakage through the needle puncture was verified following needle retraction in all samples. Histological sections of the discs displayed a clear defect at each disc's injection site with strong fluorescent labeling indicating a leakage pathway. The modeling results suggest that less than one-fourth of the injected volume was absorbed by the tissue in 4 minutes. Taken together these results suggest that needle injection is a feasible, albeit inefficient method for delivery of therapeutic agents into the intervertebral disc. Particular care should be taken to aspirate un-absorbed injectate prior to needle retraction to prevent leakage and exposure of surrounding tissues.

Detection and Separation of Inorganic Cations in Natural, Potable, and Wastewater Samples Using Capillary Zone Electrophoresis with Indirect UV Detection.

Capillary zone electrophoresis (CZE) is a sensitive and rapid technique for determining traces of inorganic cations in water samples. CZE with indirect UV-diode array detection (CZE-DAD) was utilized to identify several inorganic cations in natural, potable, and wastewater samples. A pH 4.35 background electrolyte system was employed and consisted of 15 mM imidazole, 8 mM malonic acid, 2 mM 18-crown-6 ether as complexing agents, 10% v/v methanol as an organic modifier with indirect absorbance reference at 214 nm. The CZE method involved electromigration injection at 5 kV for 5 s, a separation voltage of 20 kV at 25°C, and a detection wavelength of 280 nm. Six main cations (ammonium [Formula: see text], potassium K+, calcium Ca2+, sodium Na+, magnesium Mg2+, and lead Pb2+) were tested, and all but lead, were detected in the water samples at concentrations between 0.03 and 755 ppm with a detection limit ranging between 0.023 and 0.084 ppm. The successful evaluation of the proposed methodology allowed us to reliably detect and separate six metal ions in different water samples without any pretreatment. All water samples were collected from Northern New York towns and the Raquette River water system, the third longest river in New York State and the largest watershed of the central and western Adirondacks.

Detection and Quantification of Inorganic and Organic Anions in Natural, Potable, and Wastewaters in Northern New York Using Capillary Zone Electrophoresis and Indirect UV Detection.

Capillary zone electrophoresis (CZE) is a sensitive and rapid technique used for determining traces of inorganic and organic anions in potable, natural, and wastewaters. Here, CZE with indirect UV-diode array detection (CZE-DAD) was employed with a background electrolyte system comprising of an Agilent Technologies proprietary basic anion buffer at pH 12.0 and a forensic anion detection method. The limits of detection (LOD) for this method ranged between 3 and 5 ppm and involved hydrodynamic injection of 50 mbar for 6 s with a negative polarity separation voltage of -30 kV at 30°C, a detection wavelength of 350 nm and indirect reference of 275 nm. Fourteen different anions were checked for in the water samples that were examined and included bromide, chloride, thiosulfate, nitrate, nitrite, sulfate, azide, carbonate, fluoride, arsenate, phosphate, acetate, lactate, and silicate. The water samples were collected from Northern New York towns and the Raquette River water system, the third longest river in New York State and the largest watershed of the central and western Adirondacks. The concentrations detected for these anions ranged from <5.0 ppm to 260 ppm.