Terminal arbor degeneration--a novel lesion produced by the antineoplastic agent paclitaxel

Abstract

The antineoplastic agent paclitaxel causes a dose-limiting distal, symmetrical, sensory peripheral neuropathy that is often accompanied by a neuropathic pain syndrome. In a low-dose model of paclitaxel-evoked painful peripheral neuropathy in the rat, we have shown that the drug causes degeneration of intraepidermal nerve fibers (IENFs), i.e. the fibers which give rise to the sensory afferent's terminal receptor arbor. However, we did not find any evidence for axonal degeneration in samples taken at the mid-nerve level. Here we aimed to determine whether the absence of degenerating peripheral nerve axons was due to sampling a level that was too proximal. We used electron microscopy to study the distal-most branches of the nerves innervating the hind paw glabrous skin of normal and paclitaxel-treated rats. We confirmed that we sampled at a time when IENF degeneration was prominent. Because degeneration might be easier to detect with higher paclitaxel doses, we examined a four-fold cumulative dose range (8-32 mg/kg). We found no evidence of degeneration in the superficial subepidermal axon bundles (sSAB) that are located just a few microns below the epidermal basal lamina. Specifically, for all three dose groups there was no change in the number of sSAB per millimeter of epidermal border, no change in the number of axons per sSAB and no change in the diameter of sSAB axons. We conclude that paclitaxel produces a novel type of lesion that is restricted to the afferent axon's terminal arbor; we name this lesion 'terminal arbor degeneration'.

Fig. 1

Time course of IENF loss in animals treated with our standard protocol (8 mg/kg total dose). There is no change in the mean number of IENFs per cm of epidermal border on D7, which is the day following the last injection of paclitaxel. A statistically non-significant decrease is seen on D14, which is the approximate time of onset for paclitaxel-evoked mechano-allodynia and mechano-hyperalgesia. Statistically significant IENF decreases are seen on D22 and D27 when mechano-allodynia and mechano-hyperalgesia are at peak, or near peak, severity. * p < 0.05 vs. control group.

Fig. 2

Effects of 8 mg/kg, 24 mg/kg, and 32 mg/kg paclitaxel on the number of IENFs per cm of epidermal border. The animals were sacrificed on D29-D36. For each dose the decrease is significant relative to their vehicle-matched control group. * p < 0.05.

Fig. 3

Superficial and deep subepidermal axon bundles (SAB). sSAB were found within indentations between adjacent basal keratinocytes. The crests of such indentations were located beneath the zipper-like junction between keratinocytes (barred white arrow). dSAB were found at or just below the papillary fibroblast level. Both types of SAB are composed of unmyelinated axons wrapped in the processes of a non-myelinating Schwann cell; there is no perineurium. sSAB were often associated with a cytoplasmic process (black arrow) that traveled towards the top of the indentation; we have not been able to identify these processes. F: fibroblast; N: Nuclei of basal keratinocytes; KP: keratinocyte process which in this section appears to be separate from its cell body. Scale bar: 5.0 µm. Image from a rat treated with a cumulative dose of 32 mg/kg paclitaxel.

Fig. 4

Details of sSAB from (A) a vehicle-injected control rat and (B) a rat treated with the highest dose of paclitaxel (4 × 8 mg/kg). In both control and paclitaxel-treated rats, sSAB axons had diameters ranging from 0.3 to 1.3 µm (mean 0.45 µm) and were always unmyelinated. The axoplasm was usually pale, and sometimes watery, with scarce microtubules and neurofilaments. Thick arrows point to the epidermal basal lamina; the thin arrows point to the Schwann cell’s basal lamina. The axons below these arrows have widened mesaxons; presumably a preliminary step in the fiber leaving the bundle (see also Fig. 5C). Scale bar: 1.0 µm.

Fig. 5

Details of subepidermal axon bundles (SAB). A: A presumed dSAB to sSAB transition. The sSAB axons (black arrow), which are sectioned tangentially in this view, have left the underlying dSAB and are traveling towards an indentation between adjacent keratinocytes. Barred black arrow: unidentified process; N: Nucleus of basal keratinocyte; F: fibroblast; SC: Schwann cell nucleus. B: An sSAB lying within an indentation between adjacent keratinocytes. Thin arrow: epidermal basal lamina. Thick arrow: unidentified process; N: Nucleus of basal keratinocyte. C: Axon (asterisk) lying along the margin of an sSAB with an expanded mesaxon. Arrow: Schwann cell basal lamina. Scale bars: (A) 5.0 µm; (B) 1.0 µm; (C) 0.25 µm. All images from vehicle-treated rats.

Fig. 6

Superficial subepidermal axon bundle (sSAB) morphometry for vehicle-injected controls (Con) and rats receiving cumulative paclitaxel doses of 8, 24 and 32 mg/kg. A: Number of sSAB per mm of epidermal border. B: Number of axons per sSAB. C: Diameters of sSAB axons. Means ± SEM. There are no statistically significant differences between the control group and any of the paclitaxel dosage groups.

Fig. 7

Time course of paclitaxel-evoked mechano-allodynia (responses to 4 g von Frey hairs; VFH) and mechano-hyperalgesia (responses to 15 g VFH). Vehicle or paclitaxel were given on D0, D2, D4 and D6. B: Pre-injection baseline response frequencies. The response frequencies and time course data from the group receiving our standard cumulative dose (8 mg/kg) of paclitaxel were nearly identical to what we have seen in our prior studies. Here we have combined the current and previous 8 mg/kg data into a large N composite (10–32 rats per time point). The responses of the vehicle-injected control rats did not vary significantly over time and were also nearly identical to what we have seen in our prior studies. Thus, here we also combine the data from the current control group and the control groups from prior studies into a large N composite (10–27 rats per time point) with the dashed lines showing the response frequencies averaged over all time points tested. Note that rats receiving 8 mg/kg paclitaxel have a notable delay between the last paclitaxel injection and the appearance of mechano-allodynia and mechano-hyperalgesia (coasting) and that this delay is shorter in rats receiving 24 mg/kg and shorter still in rats receiving 32 mg/kg. * p < 0.05 relative to own baseline (Dunnett’s t-tests). Only the earliest day of significant mechano-allodynia or mechano-hyperalgesia is marked; in all cases, every day subsequent to the earliest significant time point was also significantly different from baseline with p < 0.05 or better.