See Claude et al 1982  for full method including sympathetic ganglion culture, Campenot chambers, assessment of receptor number and saturation. We used this experimental arrangement to compare transport of WGA-FITC to NGF-TR (Texas Red) to assess the impact of ATF (axonal transport facilitator) on transport. We then prepared fluorescent labeled WGA-dextran-FITC and NGF-dextran-TR for comparison in the Campenot chamber model.
By combining phage display technology with Campenot chamber technology, we were able to explore for and identify new purely synthetic ATFs. We harvested phage from the neuronal cell body after exposing the axons in sealed chambers to large numbers of phage variants. These phages were reexposed to second and third tier Campenot chamber sorting so that only well transported phage variants were selected and their surface variant proteins amplified and characterized by standard methods.
To evaluate potential clinical efficacy for analgesia in vivo, an affinity purified agent comprising WGA-dextran-gabapentin was used in a well characterized hyperalgesia model . In each of three repetitions of the experiment, groups of six rats had one of four treatments: injection of into the muscle and paw of the affected hindlimb of (1) - the full tripartite agent (WGA-dextran-drug), (2) - dextran-drug with no axonal transport facilitator or (3) - an equivalent dose of free drug or (4) - tripartite agent injected subcutaneously in the cervical fat pad for systemic delivery. Total doses of gabapentin were 75 micrograms in adult 200 gm Sprague-Dawley rats. Paw withdrawal latency in the ipsilateral and unaffected contralateral limb was measured (a total of 72 animals).
For assessment of extent of access to dorsal root ganglion (DRG) cells and to dorsal root entry zone of Lissauer regions I and II from the periphery, we injected FITC labeled WGA in biceps femoris muscle and Tetramethylrhodamine isothiocyanate (TRITC) labeled WGA in foot pad. On each of days 1-28 following injection, dorsal root ganglia were collected, sectioned and analyzed with fluorescence microscopy for counts of total cells and of cells with transported FITC. This allowed for assessment of the degree to which different cell populations could be accessed from the two different types of injection site.
To further confirm the axonal transport nerve contrast effect at appropriate time frames, we used serial solenoid coil imaging of the upper arm of a rabbit after injection of WGA-dextran-magnetite with no surgical alterations. Instead we relied on various manipulations of pulse sequence and on magnet parameters to accomplish nerve identification and to allow a bright T2 signal from nerve [, , ]. Then, by injecting the relevant musculature, we could observe a gradual decrease in the physiologically expected MRI nerve image signal.
Dorsal root entry zone access by transported agents. Section of spinal cord showing retrogradely transported NGF-TR (Nerve Growth Factor - Texas Red) in the dorsal horn. (a+b) The ipsilateral and contralateral dorsal horn viewed using darkfield microscopy. (c+d) The same fields as in a+b viewed using fluorescence microscopy. The arrow heads delineate the area of DREZ lamina I and II where the proximal axons of the dorsal root ganglia cells terminate on nociceptor neurons. Scale bars = 120 μm.
Axonal transport to spinal motor neurons and autonomic neurons. (a) Section of rat spinal cord showing retrogradely transported WGA-FITC in the motor neuron cell bodies (v) and in cells in the autonomic intermediolateral cell column (i). (b) magnified view of motor neurons seen in (a). Scale bars (a)= 120 μm, (b) = 30 μm.
Axonal transport to dorsal root ganglion neurons. (a) Delivery to rat dorsal root ganglion cells from different peripheral sources. Section of L4 dorsal root ganglia showing retrogradely transported FITC (green) injected intra-muscular and TRITC (red) injected intra-plantar, in the sensory neuron cell bodies (fp - footpad injection, ga - gastrocnemius injection) (b) higher resolution view. Scale bars (a) = 170 μm, (b) = 45 μm.
Microscopic MRI evaluation of sciatic nerve magnetite contrast effect. Microscopic MRI image of sciatic nerve in calibrating gel chamber in anaesthetized rabbit. The experimental set up is detailed in Figure 19. The wall of the silastic cuff was opened and placed surgically around the sciatic nerve. Serial images with a 2 cm surface coil allows for measurement of the relative T2 intensity of the sciatic nerve by comparison with the T2 of the calibration gels in the three surrounding chambers with the elapse of 8 hours between the pre-injection image and the post-transport image. The injection was carried out immediately following the pre-injection image. The T2 of the sciatic nerve decreases relative to the calibration chambers as axonal transport of the WGA-dextran-magnetite agent progresses. Scale bar is 4 millimeters.
WGA-dex-FITC transport to dorsal root ganglion cells. (a) Section of L4 dorsal root ganglia showing retrogradely transported FITC in the sensory neuron cell bodies. (b) The FITC can be seen in sensory dendrites arriving at the cell. Ax - axon, DRG - dorsal root ganglion, Ne - neuron cell body. Scale bars (a) = 150 μm, (b) = 80 μm.
The tripartite assemblage with [14C]-labeled gabapentin produced activity levels for gabapentin in ipsilateral neurons of more than 600 times greater than background while counts remained at background levels in contralateral neurons. This demonstrated the localizing effect of injection of polymer-bound drug conjugated to an axonal transport facilitator, and also allowed us to estimate the drug concentration achieved based on the specific activity of the [14C]-labeled gabapentin.
T2 relaxivity of hydroxide free magnetite preparations. T2 relaxivity curves for polyacrylamide "tissue" gels polymerized with uniform distributions of various dextran coated magnetite particle preparations. Relaxivity is compared with the concentration of particles in the each gel preparation as assessed by ferrozine assay of iron content after the imaging. 1/T2 was measured in a 4.7Tesla Sisco MR spectrometer. At concentrations comparable to what was achieved in nerve by axonal transport, a T2 below 30 milliseconds would be expected for any of these particle preparations. D10 = 10,000 MW dextran coating, D40 - 40,000 MW, D70 - 70,000 MW, Mgn = magnetite, WGA is wheat germ agglutinin, Seph II - sepharose separated to reduce contamination of magnetite by non-superparamagnetic ferrites.