Intraneural Drug Delivery

Molecular Synthetics intraneural drug delivery technology is based on the natural phenomenon of axonal transport. This is the mechanism that makes it possible for a neuron in the spinal cord to determine the chemical contents of its distant nerve ending in the hand or foot. In tens of thousands of academic studies, axonal transport has been used to deliver anatomical tracer compounds. Molecular Synthetics is harnessing this potential for drug delivery.

Molecular Synthetics technology includes developments related to a wide variety of compounds. In many cases, the Molecular Synthetics vehicle is able to carry dozens of molecules of a useful medication into a nerve and deliver it at high concentration in the particular location in the nervous system where it will be most effective.

Axonal Transport


Currently, medications are generally delivered through the bloodstream. This is the case whether they are administered by mouth, by intramuscular injection, or by an intravenous route. By nature ‘blood-delivered’ medications are distributed evenly through the entire body except where there are special exclusions such as the blood brain barrier.

Molecular Synthetics is developing a revolutionary class of drug delivery vehicles and medications that nerve enter the blood stream on their path to their target. Instead, these medications travel up the inside of nerves – the entirely novel ‘intraneural route.’ This is a feat that has been dreamed of by neuroscientists and pharmacologists for decades. Now, by applying its expertise and intellectual property position in nanoscale, supramolecular design, Molecular Synthetics has developed the technology that makes this dream a reality.

The resulting intraneural medications can accomplish tasks that no blood-delivered medication ever will. A single injected treatment at 1/10,000th of the oral dose can produce six days of near complete pain relief. Injection of medications of this class are expected to transform the way surgical patients are managed and also is expected to help solve many of the most intractable problems in pain management.

So, how is this remarkable technological feat accomplished? This overview is intended to provide a detailed scientific overview of the biological phenomenon of axonal transport on which these medications are based.

In a healthy nerve cell, a large fraction of all metabolic effort is expended in managing the intracellular movement of molecules and organelles. The nucleus of a motor neuron in the high lumbar terminus of the spinal cord, must manage events taking place in its synapses in e.g. a muscle in the foot nearly three feet away. The neuromuscular junction is dependent on the distant nucleus for much of its regulatory control and the nucleus needs information on chemical events taking place at the axon terminus. This remarkable intracellular informational and metabolic challenge is managed through a phenomenon called axonal transport. During the 1990’s it began to be apparent that magnetic resonance imaging could be capable of evaluating the physiology and pathology of axonal transport by the means of a novel class of intraneural contrast agents. That work led to fundamental discoveries about pharmacological aspects of axonal transport and its potential for therapeutic use.

There is strong evidence that interference with axoplasmic flow precedes interference with the transmission of electrical impulses or actual vascular compromise of the nerve.(1,2) Axonal transport continues in crushed nerves with accumulation of transported substances both proximal and distal to the site of injury(3) and so could be used to mark the location of the compression. Axonal transport is disordered in diabetic neuropathy(4,5) and amyotrophic lateral sclerosis,(6) there is at least reason to hope that clinical imaging of transport could aid in diagnosis and management. More importantly, however, it is now increasingly clear that axonal transport can be used to do the work of drug delivery for purely pharmaceutical effect. Treatable pathologies affecting the nerves, spinal cord, and the brain have typically presented severe technical challenges for the delivery of useful medications to the target sites of action. Axonal transport provides a powerful highway for transporting useful pharmaceuticals, nucleic acids, and other physiologically active compounds from easily accessible sites in the periphery to important targets behind the blood/nerve and blood/brain barrier.

There is an impressive array of compounds which have been developed for the detection of axonal transport and its sequellae in laboratory settings using agents intended only for animal experiments with histologic detection. In the 1990’s years, a number of groups(7,8,9) began to explore the development of clinically applicable analogs for the histologic tracer compounds. This effort remains at a fairly preliminary stage, but a number of promising early results have been reported.(10,11) This review is intended to lay out the cellular and biochemical background which underlies this body of work as well as to assess progress and prospects.

The work of SynGenix LTD in applying axonal transport to drug delivery to treat pain, relieve muscle spasm, and possibly to provide neuroprotection in a variety of conditions is an exciting and very recent development.

The History of Axonal Transport Studies

The slow movement of molecules along axons was first described nearly 40 years ago,(12) but with the proof of retrograde transport of proteins from axon termini in muscle back to the CNS,(13) an extensive research program began to emerge. The initial studies in adult animals were done using Evans-Blue labeled Albumin (EBA) and horseradish peroxidase (HRP).(14) At about the same time, Cowan et al(15) reported the use of radiolabelled leucine as an anterograde tracer for study of connections between nuclei in the CNS and it was subsequently demonstrated that HRP also underwent anterograde transport within the CNS.(16,17)

Neuroanatomists have since carried out thousands of studies involving the application of a tracer at one site followed 24 to 72 hours later by sacrifice of the animal, sectioning of tissue, and histologic identification of the axon termini and neuron cell bodies to which the tracers have been transported. The principal techniques used have been fluorescent studies of small dye molecules,(18,19) autoradiography of tritiated amino acids and proteins, and a variety of techniques involving HRP.

The biology of transport of HRP has been particularly well studied in the course of attempts to improve the technique. Native HRP enters damaged axon termini at injection sites, but is also taken up by “fluid phase endocytosis.” Once inside the cell, it becomes associated with vesicles that are transported towards the neuron (somatopetal transport) at rates up to 300mm/day.(20) It remains in the cell body for up to two weeks. Histologic studies use HRP as a catalyst for a peroxidase reaction to precipitate a chromogen, usually tetramethyl benzidine (TMB) as per a protocol introduced by Mesulam.(21,22)

Uptake of HRP at the cell body with subsequent anterograde transport was not demonstrated conclusively until Mesulam and Mufson(23) showed with ocular intravitreal injections that no injury to the neuron was required for uptake by retinal ganglion cells.

An improvement in sensitivity of 40 to 50 times is achieved by the binding of some plant lectins to the HRP molecule(24) with some variation in activity associated with the method used for conjugating the lectin to the HRP.(25) Selection of particular lectins can provide selective staining of different types of neurons,(26) however, the most effective and most widely studied of these is wheat germ agglutinin (WGA) derived from Triticum vulgaris. Unconjugated wheat germ agglutinin labeled with [125I] was shown to be a highly avid tracer,(27) and is actually up to 10 times more sensitive than WGA-HRP conjugates.(28,29)

Content continues below the following illustration section

Tripartite Drug Vehicles In Axonal Transport Stream: Tripartite drug vehicles include a targeting component (blue spheres) called an axonal transport facilitator (ATF), a long chain polymer such as dextran (red molecular chain), and numerous drug molecules attached to the polymer (yellow molecules). The drawing represents carriers wich actually can carry more than a hundred drug molecules in each tripartite complex. Complexes adhere to molecule in the synaptic membrane and are then carried in the axonal transport stream, along the microtubules, to reach the cell body.

Design and Assembly of Tripartite Drug Delivery Vehicles: Tripartite drug vehicles include a targeting component (blue spheres) called an axonal transport facilitator (ATF), a long chain polymer such as dextran (red molecular chain), and numerous drug molecules attached to the polymer (yellow molecules). The drawing represents carriers wich actually can carry more than a hundred drug molecules in each tripartite complex. Design of the targeting compounds is based on Molecular Synthetics breakthrough discovery technology

Delivery of Functioning Enzyme to Axoplasm Volume of Neurons: Results of early experiments on MSI technology conducted at Harvard University. These spinal cord motor neurons have been filled with an orange colored reaction product generated from an enzyme – horseradish peroxidase – that was delivered by an intraneural route. The enzyme was conjugated to wheat germ agglutinin (WGA) – a natural but non-physiological ATF (axonal transport facilitator). The complex is injected in to muscle, taken up into the nerve endings, and transported in transport vesicles. These vesicles do not destroy or digest the enzyme and it remains functional when this histological staining procedure was carried out days later. Note that the interiors of the cell bodies and their processes make up only a small fraction of the spinal cord interior.

Cross Sectional Anatomy of Nerve and Axon: A nerve is made up of several fascicles. Inside each fascicle is a large number of axons. The inside of the fascicle is protected from tbe blood stream by a blood/nerve barrier located in the perineurium. The axons them selves are wrapped inside the lipid laden Schwann cells which act as electrical insulation. The microtubules of the axonskeleton are inside the axon itself.

Nerve Components – The axoplasm inside the axon is only a small fraction of total nerve volume: The total volume of the cytoplasm of a nerve makes up only a small fraction of the total volume of a nerve. For this reason, the concentration of a drug in a nerve reflects as much two orders of magnitude greater concentration in the axoplasm itself.

Anatomy of Junction Between Nerve and Spinal Cord: A sensory neuron has its cell body in the dorsal root ganglion. One end of the neuron travels out to the periphery throught the peripheral nerve (dorsal and ventral ramus), while the other end travels into the spinal cord through the dorsal root. Inside the spinal cord, sensory neurons carrying pain signals (nociceptors) typically have a “synapse” just past the dorsal root entry zone where they pass their electrical signal on to another neuron. Other sensory neurons may continue up to the base of the brain before arriving at their first synapse. Motor neuron cell bodies are in the ventral horn of the spinal cord. The axons of these neurons travel outward through the ventral root.

Anatomy and size of spinal motor neurons: The nucleus inside the cell body of neuron must direct production of the various proteins needed by the cell. However, neurons have long axonal processes, so neurons rely on axonal transport to rapidly carry the molecules produced in the nucleus to where they are needed to do their work in the axon termins. Reverse or retrrograde transport takes place as well. A neuron can be an enormous single cell with an axon three feet in length.

Terminal expansion of an axon: An axon comes to an end at a terminal bouton. This is an enlargement where the neuron can respond to arriving electrical impulses by releasing neurotransmitter chemicals in to the synaptic space. Axonally delivered medications travel this route in reverse by entering the terminal bouton then traveling up the axon towards the cell body.

Molecular Mechanics of Axonal Transport: The axon is surrounded by the lipid laden insulation of the Schwann cells. Inside the axon there is a skeleton of microtubules. Dynein and Kinesin are energy driven motile protiens which pull molecules and vesicles along the axoskeleton and so make axonal transport take place.

Overview of Relevant Axonal Physiology

The physiology of axonal transport per se has been a very active area of research, with a literature that parallels and in some matters, overlaps the work on tracer methods carried out by neuroanatomists and histochemists and has been reviewed extensively.(30,31,32) Interest in transport includes studies on the mechanisms by which various substances enter the neuron, the rates at which various substances travel, and in the various skeletal and motile proteins involved.


The ability of neurons to engulf large intact proteins was a surprise finding that grew out of early electron microscope work, and it was immediately apparent that this process involved the formation of membrane bound vesicles.(33)

In general, cellular endocytosis of receptor-ligand complexes involves a first stage event of creation of “endosomes.” The endosomes have a highly acidic pH which tends to dissociate the ligand.(34,35) In this sequence, the receptor is then recycled back to the cell membrane for reuse while the ligand is passed along to a lysosome for degradation. It is known that this sequence can be altered for e.g. transferrin when carrying colloidal gold. In this situation, the receptor and ligand remain complexed and are passed onto the lysosome as a unit.(36) This sort of unit, packaged in the lysosome, would then undergo fast retrograde transport in the neuron such as is known for WGA-HRP.

Retrograde transport of exogenous molecules apparently involves three distinct mechanisms of intake into the cell. Fluid phase endocytosis and adsorptive endocytosis as observed with HRP and WGA-HRP respectively both result in these substances being introduced into lysosomes.(37) The third mechanism, transcytosis(38,39) is slower than normal retrograde transport, takes place in a limited compartment immediately beneath the axolemma, and differs from other mechanisms in that the receptor and ligand are not subject to lysosomal digestion during transport. [125I] WGA has been used to study this route, and it also seems to be the route taken by NGF with NGF receptor and by lofentanil bound to the opiate receptor.

Rates of Transport

It has been clear for many years that the rate of transport of a given substance is independent of electrical activity within an axon.(40) It was also appreciated early on that different classes of substances moved at different rates.(41,42)

Anterograde axonal transport has been shown to have a major fast and a slow component(43,44). The slow component is divided into “slow component a” and “slow component b” at rates of approximately 1 and 3 mm/day respectively. These slow components apparently reflect gradual structural repair and replacement of the subunits of the cytoskeleton and are not involved in the fast components important for tracer studies.(45,46)

The fast component of transport demonstrates distinct maximal rates for anterograde (300-400mm/day) and retrograde (150-300mm/day) transport. The maximal rates of transport apply to small membrane vesicles. Further, there are a variety of “waves” or distinct sets of slower transport rates exhibited in characteristic fashion by various molecules.(47,48) Rates of progression and spreading of wave fronts have been assessed using pulse labeled [35S]-protein in rat sciatic nerve subsequently excised, and “desheathed” for measurement with a linear array of proportional ß-counters.(49,50)

Cytoskeletal mechanisms

There are three major cytoskeletal elements in the axon: 1) microtubules made up of alpha and ß-tubulin monomers, 2) microfilaments composed of actin monomers, and 3) neurofilaments which are a type of intermediate filament composed of three distinct subunits.(51) A variety of studies involving intact and skinned axons with specific antibodies and various specific poisons and depolymerizing agents have proven that it is the microtubules which are the most directly involved in axonal transport.

The various cytoskeletal elements are effectively stationary in the axon.(52) Anterograde transport involves the packaging of various proteins into the membranes of transport vesicles. In anterograde transport, these vesicles and similarly sized mitochondria are pulled along the microtubules by movable bridges of “kinesin” at a characteristic rate.(53,54,55) The various slower rates of transport in anterograde motion appear to reflect molecules that either bind poorly with kinesin or that pass in and out of the vesicles during transport. Retrograde transport moves along the same microtubules but the driving protein is dynein which operates in a direction opposite to kinesin(56,57) (see figure 1).

All of this movement is ATP and calcium dependent. The metabolism involved is local, i.e., mitochondria bound to the axolemma as well as mitochondria being transported on the microtubules use glucose and oxygen absorbed through the cell membrane along the axon to generate ATP locally.

When a nerve is ligated, transport continues for many hours both proximal and distal to the ligation. Transported vesicles, proteins, and mitochondria pile up at the ligation site. Some signal which turns around and heads back to the cell body then causes anterograde fast transport to halt.(58,59) This situation continues until a critical concentration of cytoskeletal subunits accumulates via slow transport and at that time, axonal growth begins.

Physiologic Axonal Transport Facilitators

Axons will transport a tremendous variety of substances, several of which are relatively well studied. The earliest view into these was provided by studies of amino acids which are incorporated into proteins in the cell body and then transported anterograde. However, there are a variety of cell surface receptors that first bind ligands and then are endocytosed and transported anterograde or retrograde. When these endogenous ligands are conjugated to exogenous substances, the ligands act to facilitate transport of the exogenous substance via an unique and specific intraneural pathway.

Trophic Factors

The uptake and retrograde transport of Nerve Growth Factor (NGF) by adrenergic sympathetic neurons was first demonstrated by Hendry et al(60) by injection of [I125]-NGF into the anterior chamber of the eye with subsequent transport to the superior cervical ganglion. The same group also showed that NGF is transported by sensory neurons after injection into the forepaw,(61) but is not taken up or transported by motor neurons.(62) Similar studies with an antibody to dopamine ß-hydroxylase showed uptake and transport by adrenergic neurons only.(63)

Studies of the kinetics and transport capacity of NGF(64,65,66) have demonstrated that various NGF receptor molecules are responsible for the transport specificity. A monoclonal antibody (“192-IgG”) raised against an NGF receptor on pheochromocytoma cells has made it possible to show that the NGF molecule binds to the receptor protein and that the entire complex is then transported up the axon to the cell body.(67) Studies intended to demonstrate NGF localization during transport and within the cell body have shown that i.e. NGF-HRP conjugates are also endocytosed without loss of the specificity and high transport capacity of NGF.(68) Other neurotrophic factors such as CNTF (ciliary neurotrophic factor),(69) BDNF (brain derived neurotrophic factor), and NT-3 (neurotrophin-3) are transported in similar fashions but provide access to different subsets of axons.(70)

Receptors and neurotransmitters

Another interesting ligand/receptor complex involves [3H]-lofentanil and the opiate receptor which are endocytosed and transported by sensory neurons.(71,72) PET studies with [11C]-carfentanil(73,74) have been used to assess the general distribution of opiate receptors, but this approach has never been tried as a means of tracing selected tracts via axonal transport. Similar studies with GABA, D-aspartate, dopamine, norepinephrine, and serotonin have shown that uptake and transport of some neurotransmitters is a widespread phenomenon in the CNS(75,76) as is the transport of receptors.(77)

Acetylcholinesterase uptake and transport has been studied for many years because of its ease of use as a histochemical marker.(40,78) Other studies have demonstrated transport of a wide variety of substances including Vasoactive Intestinal Polypeptide (VIP),(79) cholecystokinin,(80) substance P and somatostatin,(81) and neuropeptide-Y.(82)

Non-Physiologic Transport Facilitators

The various plant lectins bind to complex carbohydrates on the cell surface of axon termini and cell bodies and, facilitate uptake by adsorptive endocytosis.(83) Most lectins have some degree of local cytotoxicity, indeed, some studies make use of the severe toxicity of lectins such as ricin and abrin to produce chromatolytic degeneration of neurons after labeling(84,85) in studies of what is called “suicide transport.” The uptake and subsequent retrograde transport of WGA in all types of peripheral neurons was first demonstrated by Stoeckel et al.(86) A variety of fluorescent small molecules such as adriamycin(87) and fluoro-gold have also been identified as subject to efficient transport.(88,89)

Binding fragments of some bacterial toxins have proven to be even more effective than WGA for increasing adsorptive specificity of conjugated HRP labeling. Both the B-chain of cholera toxin,(90,91) and the C fragment of tetanus toxin(92,93) have been used extensively.

Yet another set of studies worth mentioning involves the use of neurotrophic viruses such as Herpes Simplex,(94,95) and rabies virus(96,97) as tracers, in some cases with cDNA probes used to locate the endpoint of transport.(98)

Monoclonal Antibodies as Transport Facilitators

Using a WGA affinity chromatography column, Fabian et al(99) isolated axonal fragments that bind this lectin and used these fractions to generate polyclonal antibodies against WGA-binding glycoproteins. These antibodies, and other anti-synaptosomal Ab’s(100,101,102) were transported retrograde only. Subsequent work with monoclonal antibodies(103) identified MoAb’s subject to both anterograde and retrograde transport, with different MoAb’s following different routes of intracellular processing and transport. Various MoAb’s may also be directed to specific sets of neurons to further increase specificity of uptake.(104) Ritchie et al(106) found much greater staining of motorneurons after uptake and transport of intramuscular injections of anti-synaptosomal (polyclonal) antibodies than after WGA injections. Exogenous Ab’s administered IV or intraperitoneally are taken up by peripheral axons throughout the CNS.(105)

Transneuronal transport

Another important phenomenon is transneuronal transport wherein a transport facilitator permits a tracer to be extruded back onto the cell surface after transport thus acting to produce a sort of second injection at the next synapse in the chain.(106,107) Tetanus toxin appears to move in a specifically transsynaptic fashion,(93) but WGA and WGA-HRP are found in glia after anterograde transport of WGA-HRP,(108) and synaptic structures therefore need not be involved.

The detailed mechanisms of transneuronal transport of WGA-HRP after retrograde transport are still not completely clear, but Harrison et al(109) demonstrated that such transport did take place, and that it seemed to be enhanced by neuronal activity. Subsequent studies(110,111) have taken advantage of this phenomenon in attempts to identify interneurons involved in central pattern generators for various stereotypic movements.

Of the various transport facilitators, tetanus toxin is the most effective for “transsynaptic” labeling in which the next neuron in a synapsing series is also labeled(112) and HSV-1 has been useful for multi synapse transneuronal studies.(113,114)

Quantitative Aspects of Uptake and Transport

Margolis and LaVail(115) sought to demonstrate quantitatively the nature of anterograde transport of the WGA molecule. They performed intravitreal injections of [125I] -WGA and assessed delivery of tracer to the optic tectum. It was possible to reduce transport by 75% with excess native WGA at 1.1mM. This made it possible to calculate that the transport capacity was for 550 fmoles of WGA per 24 hours. Estimating 2.6 x 106 ganglion cells in the system, this represented 5,000 molecules of WGA transported per cell per hour. Their previous study(116) demonstrated that approximately 85% of injected [125I]-WGA arrives in the optic tectum as the same 18,000 dalton molecule injected 25 hours earlier.

Access to Transport Stream from Intra-Muscular Injection

Many studies of motorneuron organization have been carried out by the application of native HRP directly to isolated muscular branches of peripheral nerves.(117,118,119) Brushart and Mesulam(120) demonstrated the effectiveness of intramuscular and intradermal injections of WGA-HRP, attributing the effect to improved uptake and higher axon transport capacity than for native HRP. However, in an attempt to achieve distinct labeling of motorneurons in small, closely juxtaposed muscle fiber groups, it has been noted that WGA-HRP remain tightly bound at the site of injection providing impressive control of injection site specificity.(121,122,123,124) It was also found in these studies that extremely small quantities of WGA-HRP could be used without diminishing central labeling.

Injections into muscle or near sensory nerve endings work because the axon terminus presents a breach in the blood brain barrier. The perineurium, which generally is the site of the barrier in peripheral nerves, does not quite extend onto the bouton of the neuromuscular junction. Further, many axons branch extensively as they enter muscle, thus providing a relatively very large surface area which, because of the organization of motor units, may be distributed quite extensively throughout a given muscle.

Intravenous administration of tracers/transport facilitators leads to uptake at neuromuscular junctions throughout the body (125) and via brain regions which have an incomplete blood brain barrier.(126,127)

There are no quantitative studies of intramuscular uptake comparable to the anterior chamber and intravitreal studies noted above, but several aspects of the phenomenon are explained by muscle studies. In the same year that Stoeckel et al(86) discovered neuron uptake of WGA, Barchi et al(128) found that WGA had a great specificity for muscle cell membranes (sarcolemma). In addition, DeSantis and Paul(129) have shown high affinity for various parts of the muscle spindle. Local non-neuronal binding may also serve as a continuing fixed pool which continues to provide e.g. WGA for neuronal uptake for hours or days after the initial injection, thus helping to avoid limitations due to saturation of uptake receptor sites on the axon termini.

There is substantial evidence that intramuscular injections can yield high intraneural concentrations of tracer with minimal spread away from the injection site.(127) Only very recently has it become clear that the efficiency of uptake of axonal tracers after intramuscular injection is sufficient to permit consideration of clinical uses. Preliminary reports appearing during 1991 reveal that labeled proteins amenable to nuclear medicine imaging can achieve useful concentrations in nerve after intramuscular injection.(130)

Design Considerations for Intraneural Agents

Information derived from the effort to develop intraneural contrast agents for magnetic resonance provides several key findings relevant to design factors for intraneural agents in general. The bulk of MRI enhancement research in neuroimaging has involved small molecules, mostly gadolinium-DTPA, and has been based on interruptions in the blood brain barrier which permit selective enhancement of tissues involved in pathologic processes.(131) Attempts to explore the use of magnetite as a superparamagnetic enhancement agent for MRI(132,133) had been directed towards developing an intracellular agents for hepato-biliary work,(134,135) oral contrast for GI studies(136,137) and selective antibody based labels for tumors.(138,139)

An MRI tracer must have a powerful effect on proton relaxivity to be detectable in tracer quantities and gadolinium would require concentrations much greater than can be achieved intraneurally by IM injection. This is because uptake events are essentially saturable. However, a single magnetite particle can have an impact on relaxivity that is orders of magnitude greater than a single molecule of gadolinium DTPA.(140,141,142) Since the number of uptake events at the neuromuscular junction limits the amount of tracer which can be introduced into the transport stream, several groups have sought to develop magnetite based labels for axonal transport imaging, including studies with the highly effective axonal transport facilitator WGA(143) conjugated to the superparamagnetic particle. In this case, use of a ferrite contrast material with 100,000 times greater imaging effect per unit than a gadolinium agent effectively amplifies the effects of the limited number of uptake events.

Most histologic studies of axonal transport used small molecules or proteins, however, it has proven possible to use several types of particulate tracers such as colloidal gold,(144) iron dextran,(145) and fluorescent labeled latex microspheres with diameters ranging up to 200 nm.(146) Natural particulate substances in the 80-120nm size range(147) as well as larger organelles are transported(148) physiologically.

A study by Colin et al(149) with particles found that nerve injury is required to permit entrance and transport of the particles. However , the injections undertaken in that study were not intramuscular and did not involve attachment of an axonal transport facilitator. Hollander et al(150) have demonstrated uptake of latex microspheres up to 50 nm in diameter in intact cultured sympathetic neurons.

Brady(8) reported transport of MR detectable particles after direct injection into the sciatic nerve and after the completely severed sciatic nerve was soaked in a gel with ferrite particles. Ghosh et al(7) reported evidence of transport of ferrite particles after direct pressure injection into the brain of a frog although no MR detection was achieved. Enochs et al(10) report slow transport of ferrite particles after DMSO assisted intraneural injection, but could not achieve MR detectable quantities of transport with intramuscular injection.

Particles such as colloidal gold histology labels can be endocytosed and transported by nerves after intramuscular injection,(151) and it has been known for many years that dextran coated iron oxide particles are transported after intramuscular injection.(152) It has now been reported that biodegradable superparamagnetic particles, made by a modification of the Molday and MacKenzie(153) method, can also be endocytosed and transported by nerves after intramuscular injection with intraneural concentrations sufficient for detection in magnetic resonance images.(9) However, the relative concentration in spinal cord relative to nerve was much lower than was achieved with unconjugated WGA alone.(130)

Substantial amounts of some transported axonal tracers are actually deposited at nodes of Ranvier and not transported the entire distance up the nerve to the cell body(154) and there is evidence that iron dextran particles are diverted into the myelin sheath in the CNS.(155) These phenomena may account for the predominantly axonal rather than neuronal distribution and may be critical in setting a limit due to toxicity of degraded iron oxide. However, this predominantly neural rather than central distribution is preferable for the requirements of nerve imaging.

This work is relevant to broader issues affecting intraneural pharmaceuticals. It demonstrated that even large molecular complexes ranging up to 50 nm can be endocytosed and transported. Further, it demonstrates that amplification of the uptake events by delivering particles with thousands of iron atoms instead of just one gadolinium atom is an effective and desirable strategy.

Prospects for Clinical Use

Development of the imaging agents continues to be at a very preliminary stage, however, very recent advances in therapeutic drug delivery at Molecular Synthetics have now virtually assured that axonal transport will play a major role in pharmacological therapy in the new millennium.

It is now clear that small molecule drugs can be delivered in highly efficacious quantity into the interior of selected nerve groups. When pain medicines are delivered in this fashion, the result is highly effective analgesia lasting several days after a single injection using amounts of drug that are orders of magnitude less that what is required for systemic application. Intraneural drug delivery promises to usher in a new era in pharmacological therapy in which selected targets in the nervous system can be treated effectively with a wide variety of types of drugs with out the systemic and nervous system side effects which have plagued neurological pharmacology in the past.


1. Weiss DG. ‘Axoplasmic Transport.’ Berlin; Springer-Verlag 1982; pp. 477.

2. Gallant PE. The direct effects of graded axonal compression on axoplasm and fast axoplasmic transport. J Neuropathol Exp Neurol 1992; 51:220-230.

3. Bisby MA, Bulger VT. Reversal of axonal transport at a nerve crush. J Neurochem 1977; 29:313-320.

4. Tomlinson DR, Mayer JH. Defects of axonal transport in diabetes mellitus – a possible contribution to the aetiology of diabetic neuropathy. J Autonom Pharm 1984; 4:59.

5. Abbate SL, Atkinson MB, Breuer AC. Amount and speed of fast axonal transport in diabetes. Diabetes 1991; 40:111-117.

6. Munoz DG, Greene C, Perl DP, Selkoe DJ. Accumulation of phosphorylated neurofilaments in anterior horn motoneurons of amyotrophic lateral sclerosis patients. J Neuropath Exp Neurol 1988; 47:9-18.

7. Ghosh P, Zhou X, Lin W, Feng AS, Groman E, Lauterbur PC. Neuronal tracing with magnetic labels. Soc Magn Res Med Abs 1991; 10:1042.

8. Brady TJ. Future prospects for MR imaging. Soc Magn Res Med Abs 1991; 10:2.

9. Filler AG, Winn HR, Howe FA, Griffiths JR, Bell BA, Deacon TW. Axonal transport of superparamagnetic metal oxide particles: Potential for magnetic resonance assessments of axoplasmic flow in clinical neuroscience. Soc Magn Res Med Abs 1991; 10:985.

10. Enochs WS, Schaffer B, Bhide P, et al. MR Imaging of slow axonal transport. Soc Magn Res Med Abs 1992; 11:1727

11. Filler AG, Bell BA. Axonal transport, imaging, and the diagnosis of nerve. Br J Neurosurg 1992; 6:293-295.

12. Weiss PA, Hiscoe H. Experiments on the mechanism of nerve growth. J Exp Zool 1948; 107:315-396.

13. Kristensson K, Olsson Y. Retrograde axonal transport of protein. Brain Res 1971; 29:363-365.

14. Kristensson K, Olsson Y, Sjostrand J. Axonal uptake and retrograde transport of exogenous proteins in the hypoglossal nerve. Brain Res 1971; 32:399-406.

15. Cowan WM, Gottlieb DI, Hendrickson AE, Price JL, Woolsey TA. The autoradiographic demonstration of axonal connections in the central nervous system. Brain Res 1972; 37:21-51.

16. Kuypers HGJM, Kievit J, Groen-Klevant AC. Retrograde axonal transport of horseradish peroxidase in rat’s forebrain. Brain Res1974; 67:211-218.

17. Nauta HJ, Pritz MB, Lasek RJ. Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method. Brain Res 1974; 67:219-238.

18. Illert M, Fritz N, Aschoff A, Holländer H. Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat. J Neurosci Meth 1982; 6:199-218.

19. Keizer K, Kuypers HGJM, Huisman AM, Dann O. Diamidino yellow dihydrochloride (DY-2HCL); a new fluorescent retrograde neuronal tracer which migrates only very slowly out of the cell. Exp Brain Res 1983; 51:179-191.

20. Grafstein B, Forman D. Intracellular transport in neurons. Physiol Rev 1980; 60:1168-1283.

21. Mesulam M-M. Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J Hist Cyt 1978; 26:106-117.

22. Mesulam M-M. ‘Tracing Neural Connections with Horseradish Peroxidase.’ IBRO Handbook Series: Methods in the Neurosciences. Chichester; J. Wiley and Sons, 1982; pp. 251.

23. Mesulam M-M, Mufson EJ. The rapid anterograde transport of horseradish peroxidase. Neuroscience 1980; 5:1277-1286.

24. Gonatas NK, Harper C, Mizutani T, Gonatas JO. Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport. J Hist Cyt 1979; 27:728-734.

25. Trojanowski JQ, Gonatas JO, Gonatas NK. Horseradish peroxidase (HRP) conjugates of cholera toxin and lectins are more sensitive retrogradely transported markers than free HRP. Brain Res 1982; 231:33-50.

26. Borges LF, Sidman RL. Axonal transport of lectins in the peripheral nervous system. J Neurosci 1982; 2:647-653.

27. Schwab ME, Javoy-Agid F, Agid Y. Labeled wheat germ agglutinin (WGA) as a new highly sensitive retrograde tracer in the rat brain hippocampal system. Brain Res 1978; 152:145-150.

28. Rhodes CH, Gonatas JO, Gonatas NK. A quantitative comparison of the efficiency of orthograde axonal transport and transsynaptic transport of iodinated [125I] wheat germ agglutinin (I-WGA) and horseradish peroxidase labeled I-WGA (I-WGA-HRP) in the rat visual system. Brain Res 1985; 336:376-380.

29. Trojanowski JQ. Native and derivatized lectins for in vivo studies of neuronal connectivity and neuronal cell biology. J Neurosci Meth 1983; 9:185-204.

30. Ochs S. A brief history and present status of transport mechanism models. In: R. S. Smith and M. A. Bisby Eds. – “Axonal Transport,” New York; Alan R. Liss 1987. Neurology and Neurobiology 1987; 25:1-14.

31. Schmitt FO. Introduction: Historic Context. In: The role of fast transport in the nervous system. Neurosci. Res. Prog. Bull. Vol 1981; 20 (1).

32. Smith RS, Snyder RE. Relationships between the rapid axonal transport of newly synthesized proteins and membranous organelles. Mol Neurobiol 1992; 6:285-300.

33. Rosenbluth J, Wissig SL. The distribution of exogenous ferritin in toad spinal ganglia and the mechanism of its uptake by neurons. J Cell Biol 1964; 23:307-325.

34. Stahl P, Schwartz AL. Receptor-mediated endocytosis. J Clin Inv 1985; 77:657-662.

35. Schmid SL, Fuchs R, Male P, Mellman I. Two distinct subpopulations of endosomes involved in membrane recycling and transport of lysosomes. Cell 1988; 52:73-83.

36. Neutra MR, Ciechanover A, Owen LS, Lodish HF. Intracellular transport of transferrin- and asialloorosomucoid-colloidal gold conjugates to lysosomes after receptor mediated endocytosis. J Hist Cyt 1985; 33:1134-1144.

37. Harper C, Gonatas JO, Steiber A, Gonatas NK. In vivo uptake of wheat germ agglutinin-horseradish peroxidase conjugates into neuronal GERL and lysosomes. Brain Res 1980; 188:465-472.

38. Mostov KE, Simister NE. Transcytosis. Cell 1985; 43:389-390.

39. LaVail JH, Margolis TP. The anterograde axonal transport of wheat germ agglutinin as a model for transcellular transport in neurons. In: R. S. Smith and M. A. Bisby Eds. – “Axonal Transport,” New York; Alan R. Liss 1987. Neurology and Neurobiology 1987; 25:311-326.

40. Jankowska E, Lubinska L, Niemierko S. Translocation of AChE-containing particles in the axoplasm during nerve activity. Comp Biochem Physiol 1969; 28:907-913.

41. Lasek RJ. Axoplasmic transport of labeled proteins in rat ventral motoneurons. Exp Neurol 1968; 21:41-51.

42. Ochs S. Fast transport of materials in mammalian nerve fibers. Science 1972; 176:252-260.

43. Sjöstrand J. Fast and slow components of axoplasmic transport in the hypoglossal and vagus nerves of the rabbit. Brain Res 1970; 18:461-467.

44. Willard M, Cowan WM, Vagelos PR. The polypeptide composition of intra-axonally transported proteins: evidence for four transport velocities. Proc Natl Acad Sci USA 1974; 71:2183-2187.

45. McQuarrie IG, Brady ST, Lasek RJ. Diversity in the axonal transport of structural proteins: major differences between optic and spinal axons in the rat. J Neurosci 1986; 6:1593-1605.

46. Nixon RA. Slow axonal transport. Curr Opin Cell Biol 1992; 4:8-14.

47. Lasek RJ, Brady ST. The axon: a prototype for studying expressional cytoplasm. Cold Spr Hrb Symp Quant Biol 1982; 46:113-123.

48. Baitinger C, Willard M. Axonal transport of synapsin I-like proteins in rabbit retinal ganglion cells. J Neurosci 1987; 7:3723-3735.

49. O’brien DW, Snyder RE. Position sensitive studies of the axonal transport of a pulse of radioisotope. J Neurobiol 1982; 13:435-445.

50. Snyder RE, Smith RS. Physical methods for the study of the dynamics of axonal transport. CRC Crit Rev Bioeng 1985; 10:89-123.

51. Nixon RA. The axonal transport of cytoskeletal proteins: a reappraisal.In: R. S. Smith and M. A. Bisby Eds. – “Axonal Transport,” New York; Alan R. Liss 1987. Neurology and Neurobiology 1987; 25:175-200.

52. Nixon RA, Logvinenko KB. Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons. J Cell Biol 1986; 102:647-659.

53. Schnapp BJ, Reese TS. New developments in understanding rapid axonal transport. Trends Neurosci 1986; 9:155-162.

54. Vale RD, Reese TS, Sheetz MP. Identification of a novel force-generating protein, Kinesin, involved in microtubule-based motility. Cell 1985; 42:39-50.

55. Cyr JL, Brady ST. Molecular motors in axonal transport. Cellular and molecular biology of kinesin. Mol Neurobiol 1992; 6:137-155.

56. Schnapp BJ, Reese TS. Dynein is the motor for retrograde axonal transport of organelles. Proc Natl Acad Sci USA 1989; 86:1548-1552.

57. Vallee RB, Bloom GS. Mechanisms of fast and slow axonal transport. Ann Rev Neurosci 1991; 14:59-92.

58. Bulger VT, Bisby MA. Reversal of axonal transport in regenerating nerves. J Neurochem 1978; 31:1411-1418.

59. Snyder RE. The design and construction of a multiple proportional counter used to study axonal transport. J Neurosci Meth 1986; 11:79-88.

60. Hendry IA, Stöckel K, Thoenen H, Iversen LL. The retrograde axonal transport of nerve growth factor. Brain Res 1974; 68:103-121.

61. Stoeckel K, Schwab M, Thoenen H. Specificity of retrograde transport of nerve growth factor (NGF) in sensory neurons: a biochemical and morphological study. Brain Res 1975; 89:1-14.

62. Stoeckel K, Schwab M, Thoenen H. Comparison between the retrograde axonal transport of nerve growth factor and tetanus toxin in motor, sensory, and adrenergic neurons. Brain Res 1975; 99:1-16.

63. Fillenz M, Gagnon C, Stoeckel K, Thoenen H. Selective uptake and retrograde axonal transport of dopamine-ß-hydroxylase antibodies in peripheral adrenergic neurons. Brain Res 1976; 114:293-303.

64. Johnson EM, Andres RY, Bradshaw RA. Characterization of the retrograde transport of nerve growth factor (NGF) using high specific activity [125I]-NGF. Brain Res 1978; 150:319-331.

65. Sutter A, Riopelle RJ, Harris-Warrick RM, Shooter EM. Nerve growth factor receptors: characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells. J Biol Chem 1979; 254:5972-5982.

66. Stach RW, Lyons CR, Perez-Polo JR. Characteristics of partially purified nerve growth factor receptor. J Neurochem 1987; 49:1280-1285.

67. Johnson EM, Taniuchi M, Clark HB et al. Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system. J Neurosci 1987; 7:923-929.

68. Schwab ME. Ultrastructural localization of a nerve growth factor-horseradish peroxidase (NGF-HRP) coupling product after retrograde axonal transport in adrenergic neurons. Brain Res 1977; 130:190-196.

69. Curtis R, Adryan KM, Zhu Y, Harkness PJ, Lindsay RM, DiStefano PS. Retrograde axonal transport of ciliary neurotrophic factor is increased by peripheral nerve injury. Nature 1993; 365:253-255.

70. DiStefano PS, Friedman B, Radziejewski C, et al. The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron 1992; 8:983-993.

71. Laduron PM, Janssen PFM. Axoplasmic transport and possible recycling of opiate receptors labelled with [3H]-lofentanil. Life Sci 1982; 31:457-462.

72. Laduron PM, Janssen PFM. Retrograde axonal transport of receptor-bound opiate in the vagus and delayed accumulation in the nodose ganglion. Brain Res 1985; 333:389-392.

73. Wagner HN. Radiolabeled drugs as probes of central nervous system neurons. Clin Chem 1985; 31:1521-1524.

74. Frost JJ, Wagner HN, Dannals RF. Imaging opiate receptors in the human brain by positron tomography. J Comp Assist Tom 1985; 9:231-236.

75. Streit P. Selective retrograde labeling indicating the transmitter of neuronal pathways. J Comp Neur 1980; 191:429-463.

76. Gaudin-Chazal G, Seyfritz N, Araneda S, Vigier D, Puizillout J-J. Selective retrograde transport of [3H]-serotonin in vagal afferents. Brain Res Bull 1982; 8:503-509.

77. Laduron P. Axoplasmic transport of muscarinic receptors. Nature 1980; 286:287-288.

78. Wooten GF, Cheng C-M. Transport and turnover of acetylcholinesterase and choline acetyltransferase in rat sciatic nerve and skeletal muscle. J Neurochem 1980; 34:359-366.

79. Lundberg JM, Fahrenkrug J, Brimijoin S. Characteristics of the axonal transport of vasoactive intestinal polypeptide (VIP) in nerves of the cat. Acta Phys Scand 1981; 112:427-436.

80. Rehfeld JF, Lundberg JM. Cholecystokinin in feline vagal and sciatic nerves: concentration, molecular form and transport velocity. Brain Res 1983; 275:341-347.

81. Gilbert RFT, Emson PC, Fahrenkrug J, Lee CM, Penman E, Wass J. Axonal transport of neuropeptides in the cervical vagus nerve of the rat. J Neurochem 1980; 34:108-113.

82. Fried G, Lundberg JM, Theodorsson-Norheim E. Subcellular storage and axonal transport of neuropeptide Y (NPY) in relation to catecholamines in the cat. Acta Phys Scand 1985; 125:145-154.

83. Trojanowski JQ, Gonatas JO, Gonatas NK. Conjugates of horseradish peroxidase (HRP) with cholera toxin and wheat germ agglutinin are superior to free HRP as orthogradely transported markers. Brain Res 1981; 223:381-385.

84. Olsnes S, Refsnes K, Pihl A. Mechanism of action of the toxic lectins abrin and ricin. Nature 1974; 249:627-631.

85. Wiley RG, Blessing WW, Reis DJ. Suicide transport: destruction of neurons by retrograde transport of ricin, abrin, and modeccin. 1982; Science 216: 889-890.

86. Stoeckel K, Schwab M, Thoenen H. Role of gangliosides in the uptake and retrograde transport of cholera and tetanus toxin as compared to nerve growth factor and wheat germ agglutinin. Brain Res 1977; 132:273-285.

87. Bigotte L, Olsson Y. Retrograde transport of doxorubicin (adriamycin) in peripheral nerves of mice. Neurosci Lett 1982; 32:217-221.

88. Schmued LC, Fallon JH. Fluoro-Gold: a new fluorescent retrograde axonal tracer with numerous unique properties. Brain Res 1986; 377:147-154.

89. Naso WB, Cox RD, McBryde JP, Perot PL. Rubrospinal neurons and retrograde transport of fluoro-gold in acute spinal cord injury–a dose-response curve. Neurosci Lett 1993; 155:125-127.

90. McIlhinney RAJ, Bacon SJ, Smith AD. A simple and rapid method for the production of cholera B-chain coupled to horseradish peroxidase for neuronal tracing. J Neurosci Meth 1988; 22:189-194.

91. Rivero-Meli’an C, Grant G. Choleragenoid horseradish peroxidase used for studying projections of some hindlimb cutaneous nerves and plantar foot afferents to the dorsal horn and Clarke’s column in the rat. Exp Brain Res 1991; 84:125-32.

92. Schwab ME, Suda K, Thoenen H. Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal transport. J Cell Biol 1979; 82:798-810.

93. Fishman PS, Savitt JM, Carrigan DR. Transsynaptic transfer of the binding fragment of tetanus toxin. Soc Neurosci Abs 1987; 13:121.

94. Esiri MM, Tomlinson AH. 1984; Herpes simplex encephalitis: Immunohistological demonstration of spread of virus via olfactory and trigeminal pathways after infection of facial skin of mice. J Neurol Sci 1984; 64:213-217.

95. Kuypers HGJM, Ugolini G. Viruses as transneuronal tracers. TINS 1990; 13:71-75

96. Tsiang H, Ceccaldi PE, Lycke E. Rabies virus infection and transport in human sensory dorsal root ganglia neurons. J Gen Virol 1991; 72:1191-1194.

97. Astic L, Saucier D, Coulon P, Lafay F, Flamand A. The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice. Brain Res 1993; 619:146-156.

98. Ermine A, Ceccaldi PE, Masson G, Tsiang H. Rabies RNA synthesis, detected with cDNA probes, as a marker for virus transport in the rat nervous system. Mol Cell Probes 1993; 7:1-5.

99. Fabian RH, Ritchie TC, Coulter JD. Properties of axonally transported antibodies to rat brain membrane fractions. Soc Neurosc Abs 1984; 10:353.

100. Wenthold RJ, Skaggs KK, Reale RR. Retrograde axonal transport of antibodies to synaptic membrane components. Brain Res 1984; 304:162-165.

101. iWenthold RJ, Skaggs KK, Reale RR. Characterization of retrograde axonal transport of antibodies in central and peripheral neurons. J Hist Cyt 1986; 34:373-380.

102. Ritchie TC, Fabian RH, Coulter JD. Axonal transport of antibodies to subcellular and protein fractions of rat brain. Brain Res1985; 343:252-261.

103. Ritchie TC, Fabian RH, Choate JV, Coulter JD. Axonal transport of monoclonal antibodies. J Neurosci 1986; 6:1177-1184.

104. Hinton DR, Henderson VW, Blanks JC, Rudnicka M, Miller CA. Monoclonal antibodies react with neuronal subpopulations in the human nervous system. J Comp Neur 1988; 267:398-408.

105. Fabian RH, Petroff G. Intraneuronal IgG in the central nervous system: uptake by retrograde axonal transport. Neurology 1987; 37:1780-1784.

106. Gerfen R, O’Leary DDM, Cowan WM. A note on the transneuronal transport of wheat germ agglutinin-conjugated horseradish peroxidase in the avian and rodent visual systems. Exp Brain Res 1982; 48:443-448.

107. Ruda M, Coulter JD. Axonal and transneuronal transport of wheat germ agglutinin demonstrated by immunocytochemistry. Brain Res 1982; 249:237-246.

108. Rhodes CH, Stieber A, Gonatas NK. Transneuronally transported wheat germ agglutinin labels glia as well as neurons in the rat visual system. J Comp Neur 1987; 261:460-465.

109. Harrison PJ, Hultborn H, Jankowska E, Katz R, Storai B, Zytnicki D. Labelling of interneurones by retrograde transsynaptic transport of horseradish peroxidase from motoneurones in rats and cats. Neurosci Lett 1984; 45:15-19.

110. Jankowska E, Skoog B. Labelling of midlumbar neurones projecting to cat hindlimb motoneurones by transneuronal transport of a horseradish peroxidase conjugate. Neurosci Lett 1986; 71:163-168.

111. Alstermark B, Kümmel H. Transneuronal labelling of neurones projecting to forelimb motoneurones in cats performing different movements. Brain Res 1986; 376:387-391.

112. Cabot JB, Mennone A, Bogan N, Carroll J, Evinger C, Erichsen JT. Retrograde, trans-synaptic and transneuronal transport of fragment C of tetanus toxin by sympathetic preganglionic neurons. Neuroscience 1991; 40:805-823.

113. Zemanick MC, Strick PL, Dix RD. Direction of transneuronal transport of herpes simplex virus 1 in the primate motor system is strain-dependent. Proc Natl Acad Sci USA 1991; 88:8048-8051

114. Ding ZQ, Li YW, Wesselingh SL, Blessing WW. Transneuronal labelling of neurons in rabbit brain after injection of herpes simplex virus type 1 into the renal nerve. J Auton Nerv Syst 1993; 42:23-31.

115. Margolis TP, LaVail JH. Further evidence in support of the selective uptake and anterograde transport of [125I]-wheat germ agglutinin by chick retinal ganglion cells. Brain Res 1984; 324:21-27.

116. Margolis TP, Marchand CM-F, Kistler HB, LaVail JH. Uptake and anterograde axonal transport of wheat germ agglutinin from retina to optic tectum in the chick. J Cell Biol 1981; 89:152-156.

117. Ruigrok TJH, Crowe A. The organization of motoneurons in the turtle lumbar spinal cord. J Comp Neur 1984; 228:24-37.

118. Smith CL, Hollyday M. The development and postnatal organization of motor nuclei in the rat thoracic spinal cord. J Comp Neur 1983; 220:16-28.

119. Fetcho JR. The organization of the motoneurons innervating the axial musculature of vertebrates. II. Florida water snakes (Nerodia fasciata pictiventris). J Comp Neur 1986; 249:551-563.

120. Brushart TM, Mesulam M-M. Transganglionic demonstration of central sensory projections from skin and muscle with HRP-lectin conjugates. Neurosci Lett 1980; 17:1-6.

121. Sokoloff A. Musculotopic organization of the hypoglossal nucleus in the grass frog (Rana pipiens). Soc Neurosci Abs 1987; 13:305.

122. Sokoloff AJ, Deacon TW. Musculotopic organization of the hypoglossal nucleus in the cynomolgus monkey, Macaca fascicularis. J Comp Neurol 1992; 324:81-93.

123. Kramer M, Deacon T, Sokoloff A, Filler A. Organization of motoneurons innervating epaxial and hypaxial musculature in the frog, rat, and monkey. Soc Neurosci Abs 1987; 13:526.

124. Hardman VJ, Brown MC. Accuracy of reinnervation of rat intercostal muscles by their own segmental nerves. J Neurosci 1987; 7:1031-1036.

125. Kristensson K. Retrograde axonal transport of horseradish peroxidase: Uptake at mouse neuromuscular junctions following systemic injection. Acta Neuropath 1977; 38:143-147.

126. Weiss ML, Cobbett P. Intravenous injection of Evans Blue labels magnocellular neuroendocrine cells of the rat supraoptic nucleus in situ and after dissociation. Neuroscience 1992; 48:383-95.

127. Merchenthaler I. Neurons with access to the general circulation in the central nervous system of the rat: a retrograde tracing study with fluoro-gold. Neuroscience 1991; 44:655-662.

128. Barchi RL, Bonilla E, Wong M. Isolation and characterization of muscle membranes using surface-specific labels. Proc. Natl. Acad. Sci. USA 1977; 74:34-38.

129. DeSantis M, Paul J. The affinity of concanavalin A and wheat germ agglutinin for components of the muscle spindle. Histochem 1979; 60:225-230.

130. Filler AG, Winn HR, Westrum LE, Sirrotta P, Krohn K, and Deacon TW. Intramuscular injection of WGA yields systemic distribution adequate for imaging of axonal transport in intact animals. Soc Neurosci Abs 1991; 17:1480.

131. Wolf, G.L. (1989) – Current status of MR Imaging contrast agents: Special report. Radiology 172:709-710.

132. Olsson M, Persson BRB, Salford LG, Schroder U. Ferromagnetic particles as contrast agent in T2 NMR-imaging. 4th Annual Meeting, Soc. Magn. Reson Med Abs 1985; 889-890.

133. Fahlvik AK, Klaveness J, Stark DD. Iron oxides as MR imaging contrast agents. J Magn Reson Imaging 1993; 3:187-194.

134. Weissleder, R., D.D. Stark, C.C. Compton, J. Wittenberg, and J.T. Ferrucci (1987) – Ferrite-enhance MR imaging of hepatic lymphoma: An experimental study in rats. AJR 149:1161-1165.

135. Saini, S., D.D. Stark, P. F. Hahn, J.-C. Bousquet, J. Introcasso, J. Wittenberg, T.J. Brady, and J.T. Ferruci (1987) – Ferrite particles: A superparamagnetic MR contrast agent for enhanced detection of liver carcinoma. Radiology 162:217-222.

136. Widder, D.J., R.R. Edelman, W.L. Grief, and L. Monda (1987) – Magnetite albumin suspension: A superparamagnetic oral MR contrast agent. AJR 149:839-843.

137. Lönnemark, M., A. Hemmingsson, T. Bach-Gansmo, A. Ericsson, A. Öksendal, R. Nyman, and A. Moxnes. (1989) – Effect of superparamagnetic particles as oral contrast medium at magnetic resonance imaging. A phase I clinical study. Acta Radiologica 30:193 – 196.

138. Cerdan, S., H. R. Lötshcer, B. Künnecke, and J. Seelig (1989) -Monoclonal antibody-coated magnetite particles as contrast agents in magnetic resonance imaging of tumors. Magnetic Resonance in Medicine 12:151-163.

139. Renshaw, P.F., C.S. Owen, A.E. Evans, and J.S. Leigh (1986) – Immunospecific NMR Contrast Agents. Magnetic Resonance Imaging 4:351-357.

140. Majumdar, S., S. Zoghbi, C.R. Pope, and J.C. Gore (1989) – A quantitative study of relaxation rate enhancement produced by iron oxide particles in polyacrylamide gels and tissue. Magnetic Resonance in Medicine 9:185-202.

141. Majumdar, S., S.S. Zoghbi, and J.C. Gore (1989) – The influence of pulse sequence on the relaxation effects of superparamagnetic iron oxide contrast agents. Magnetic Resonance in Medicine 10:289-301.

142. Josephson, L., J. Lewis, P. Jacobs, P.F. Hahn, and D.D. Stark (1988) – The effects of iron oxides on proton relaxivity. Magnetic Resonance Imaging 6:647-653.

143. Steiber, A., S.D. Erulkar, and N.K. Gonatas (1989) – A hypothesis for the superior sensitivity of wheat germ agglutinin as a neuroanatomical probe. Brain Res. 495:131-139.

144. Quattrochi J, Madison R, Sidman R, and Kijavin I. Colloidal gold fluorescent microspheres: A new retrograde maker visualized by light and electron microscopy. Exp Neurol 1987; 96:219-224.

145. Nguyen-LeGros J, Cesaro P, Gay M, Pollin B. Evolution du lieu de stockage du dextran-fer transporté par le flux axonal rétrograde dans le système nerveux central du rat. Acta Neuropath 1981; 54:101-112.

146. Katz LC, Burkhalter A, Dreyer WJ. Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex. Nature 1984; 310:498-500.

147. Studelska DR, Brimijoin S. Partial isolation of two classes of dopamine beta-hydroxylase-containing particles undergoing rapid axonal transport in rat sciatic nerve. J Neurochem 1989; 53:622-31.

148. Dahlsrom AB, Czernik AJ, Li JY. Organelles in fast axonal transport. What molecules do they carry in anterograde vs retrograde directions, as observed in mammalian systems? Mol Neurobiol 1992; 6:157-177.

149. Colin W, Donoff RB, Foote WE. Fluorescent latex microspheres as a retrograde tracer in the peripheral nervous system. Brain Res 1989; 486:334-339.

150. Hollander H, Egensperger R, Dirlich G. Size distribution of rhodamine-labelled microspheres retrogradely transported in cultured neurons. J Neurosci Meth 1989; 29:1-4.

151. Philippe E, Droz B. Calbindin-immunoreactive sensory neurons of dorsal root ganglion project to skeletal muscle in the chick. J Comp Neur 1989; 283:153-160.

152. Olsson T, Kristensson K. A simple histochemical method for double labelling of neurons by retrograde axonal transport. Neurosci Lett 1978; 8:265-268.

153. Molday RS, Mackenzie D. Immunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells. J Immunol Meth 1982; 52:353-367.

154. Berthold C-H, Mellström A. Peroxidase activity at consecutive nodes of Ranvier in the nerve to the medial gastrocnemius muscle after intramuscular administration of horseradish peroxidase. Neuroscience 1986; 19:1349-1362.

155. Nguyen-LeGros J, Cesaro P, Pollin B, Raoux N. Impaired transport of iron-dextran complex in myelinated central fibers. Neurosci Res 1985; 3:71-78.