An evolving cellular pathology occurs in dorsal root ganglia, peripheral nerve and spinal cord following intravenous administration of paclitaxel in the rat

Abstract

Paclitaxel (Taxol) is a frontline antineoplastic agent used to treat a variety of solid tumors including breast, ovarian, or lung cancer. The major dose limiting side effect of paclitaxel is a peripheral sensory neuropathy that can last days to a lifetime. To begin to understand the cellular events that contribute to this neuropathy, we examined a marker of cell injury/regeneration (activating transcription factor 3; ATF3), macrophage hyperplasia/hypertrophy; satellite cell hypertrophy in the dorsal root ganglia (DRG) and sciatic nerve as well as astrocyte and microglial activation within the spinal cord at 1, 4, 6 and 10 days following intravenous infusion of therapeutically relevant doses of paclitaxel. At day 1 post-infusion, there was an up-regulation of ATF3 in a subpopulation of large and small DRG neurons and this up-regulation was present through day 10. In contrast, hypertrophy of DRG satellite cells, hypertrophy and hyperplasia of CD68(+) macrophages in the DRG and sciatic nerve, ATF3 expression in S100beta(+) Schwann cells and increased expression of the microglial marker (CD11b) and the astrocyte marker glial fibrillary acidic protein (GFAP) in the spinal cord were not observed until day 6 post-infusion. The present results demonstrate that using the time points and markers examined, DRG neurons show the first sign of injury which is followed days later by other neuropathological changes in the DRG, peripheral nerve and dorsal horn of the spinal cord. Understanding the cellular changes that generate and maintain this neuropathy may allow the development of mechanism-based therapies to attenuate or block this frequently painful and debilitating condition.

Figures

Figure 1

Activating transcription factor 3 immunoreactivity (ATF3-IR) is increased in neurons and non-neuronal satellite cells in L4 dorsal root ganglia (DRG) of rats following intravenous paclitaxel administration. L4 DRG section from paclitaxel (C-F) and vehicle (cremephor/ethanol) treated rats (A,B) were examined immunohistochemically with antibodies against activating transcription factor (ATF3, red) a marker of cellular injury/regeneration at several time points following administration of paclitaxel. Sensory neurons in DRG were identified immunohistochemically with antibodies against NeuN (NeuN, blue) a neuronal nuclei marker (A) that labels both small (small arrow) and large (large arrow) diameter sensory neurons. Increased ATF3 immunoreactivity (IR) was observed in neuronal nuclei at day 4 (C) day 6 (D) and to a lesser extent at day 10 post paclitaxel administration (E). At day 10, there was also an increase in clusters of ATF3-IR satellite cells (E, arrowhead) within the DRG of paclitaxel-treated rats as these cells coexpressed the intermediate filament glial fibrillary acidic protein (F, GFAP, green). No ATF3 expression was observed in L4 DRG of vehicle-treated rats at day 10 (B) or other time points examined. Scale bar: A-E 30 μm. F: 25 μm.

Figure 2

Glial fibrillary acidic protein immunoreactivity (GFAP-IR) is increased in satellite cells surrounding sensory neurons in the L4 dorsal root ganglia (DRG) of rats following intravenous paclitaxel administration. L4 DRG sections from vehicle (cremephor/ethanol) (A) and paclitaxel-treated rats (B-D) were examined immunohistochemically with an antibody against GFAP, an intermediate filament present in DRG satellite cells. Increased GFAP-IR was observed at day 6 (C) and day 10 (D) following paclitaxel administration. DRG sections from paclitaxel treated rats at day 4 (B) or earlier time points displayed similar levels of GFAP-IR compared to vehicle-treated rats (A). Scale bars A-D = 30 μm.

Figure 3

The number of CD68 immunoreactive (IR) macrophages increased in L4 dorsal root ganglia (DRG) following intravenous paclitaxel administration. L4 DRG sections from vehicle (cremephor/ethanol) (A) and paclitaxel-treated rats (B-D) were examined immunohistochemically with an antibody against CD68 (clone ED1), an intracellular lysosomal protein that is expressed in activated macrophages. The number of CD68-IR macrophages increased in L4 DRG of paclitaxel treated rats beginning on day 6 through day 10. DRG sections from paclitaxel-treated rats day 4 (B) or earlier time points displayed similar numbers of CD68-IR macrophages compared to vehicle-treated rats (A). Note that CD68-IR macrophages in paclitaxel-treated rats were larger in size and formed aggregates around neuronal cell bodies (D). CD68-IR macrophages in vehicle-treated rats (C) were evenly dispersed throughout the DRG and were morphologically long and slender in appearance. Scale bars: A-D = 30 μm.

Figure 4

Activating transcription factor 3 (ATF3) is upregulated in Schwann cells and the number of CD68-IR macrophages increases in the sciatic nerve of rats following paclitaxel administration. Sciatic nerve sections from midthigh level were immunohistochemically labeled with antibodies against S100β (blue) which labels the outermost portion of myelinating Schwann cells and ATF3 (red). The number of ATF3-IR S100β-IR within the sciatic nerve of paclitaxel-treated rats increased at day six (C) and day 10 (E) following paclitaxel administration compared to vehicle-treated rats (A). Note that Schwann cells expressing ATF-3 appeared to be grouped in a linear arrangement (arrowheads) as if a group of Schwann cells that ensheath the same injured axon upregulated ATF3. Similarly, the number and size of CD68-IR macrophages was greater in the sciatic nerve of paclitaxel-treated rats at day 10 (F) compared to vehicle-treated rats (B). The number of CD68-IR macrophages in the sciatic nerve of paclitaxel-treated rats at day 6 (D) and earlier time points was similar compared to vehicle-treated rats (B) Scale bars A-F = 20 μm.

Figure 5

CD11b immunoreactivity (CD11b-IR) is increased in microglia within the dorsal horn of the L4 spinal cord of paclitaxel-treated rats. Microglia in the spinal cord of vehicle (cremophor/ethanol) treated rats (A) are uniformly distributed throughout the gray matter. Beginning six and ten days (B) following initial intravenous administration of paclitaxel an increase in CD11b-IR microglia (CD11b) is present in the dorsal horn of the spinal cord (B). Note that the increased CD11b-IR is especially prominent in deeper laminae (III-VI) of paclitaxel-treated rats. Higher magnification shows increased density of microglia with hypertrophic morphology within the spinal cord of paclitaxel-treated rats (D), whereas microglia in vehicle-treated rats possess a more ramified appearance typical of resting microglia (C). Scale bar A, B = 200μm; C, D = 30μm.

Figure 6

Glial fibrillary acidic protein immunoreactivity (GFAP-IR) is increased in astrocytes within the L4 spinal cord of paclitaxel-treated rats. Beginning six and ten days (B) following intravenous administration of paclitaxel an increase in GFAP-IR is present in astrocytes throughout the dorsal horn (laminae I-VI) of the L4 spinal cord of paclitaxel-treated rats (B) compared to vehicle (cremophor/ethanol) treated rats (A). Higher magnification demonstrates that astrocytes in paclitaxel treated rats posses a more hypertrophic appearance typical of activated astrocytes (D) compared to vehicle-treated rats (C). Scale bar A, B = 200μm; C, D = 30μm.

Figure 7

Schematic diagram depicting a primary afferent neuron and supporting cells under normal conditions and following treatment with the chemotherapeutic agent paclitaxel. a) Normally sensory neuron cell bodies within the dorsal root ganglia (DRG) are surrounded by several satellite cells that maintain neuronal homeostasis by regulating extracellular ion concentrations and nutrient levels. Resident macrophages survey the local environment for signs of tissue injury or infection. Peripheral nerve axons are surrounded by Schwann cells and project to the spinal cord and peripheral tissues. Sequelae of cellular pathology occurs in the DRG, sciatic nerve, and spinal cord following repeat (Day 0 and 3) intravenous administration of paclitaxel. B) Four days post initial infusion a subset of DRG sensory neurons upregulate activating transcription factor 3 (ATF3, red nuclei) and exhibit displaced nuclei indicative of an injured phenotype. C) Six days post initial infusion there is activation of DRG satellite cells evident as hypertrophy and increased immunoreactivity for glial fibrillary acidic protein (GFAP) as well as an increase in the number of activated (CD68-IR) macrophages in the DRG. Both microglial and astrocyte activation is present in the spinal cord beginning six days post initial infusion. D) Ten days post initial infusion nodules of Nageotte form in the DRG evident as clusters of ATF3-IR satellite cells. This feature has been suggested to indicate degeneration and loss of neuronal cell bodies. The number of CD68-IR macrophages and the expression of ATF3 in Schwann cells is increased within the sciatic nerve at day 10. Collectively, these changes in the DRG, peripheral nerves and spinal cord may be involved in the positive painful symptoms such as pain, cold allodynia, and myalgias seen in patients treated with paclitaxel as well as sensory deficits including loss of two-point discrimination, vibratory sense, and proprioceptive abilities.