Characterisation of Immune and Neuroinflammatory Changes Associated with Chemotherapy-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) and associated neuropathic pain is a debilitating adverse effect of cancer treatment. Current understanding of the mechanisms underpinning CIPN is limited and there are no effective treatment strategies. In this study, we treated male C57BL/6J mice with 4 cycles of either Paclitaxel (PTX) or Oxaliplatin (OXA) over a week and tested pain hypersensitivity and changes in peripheral immune responses and neuroinflammation on days 7 and 13 post 1st injection. We found that both PTX and OXA caused significant mechanical allodynia. In the periphery, PTX and OXA significantly increased circulating CD4+ and CD8+ T-cell populations. OXA caused a significant increase in the percentage of interleukin-4+ lymphocytes in the spleen and significant down-regulation of regulatory T (T-reg) cells in the inguinal lymph nodes. However, conditional depletion of T-reg cells in OXA-treated transgenic DEREG mice had no additional effect on pain sensitivity. Furthermore, there was no leukocyte infiltration into the nervous system of OXA- or PTX-treated mice. In the peripheral nervous system, PTX induced expression of the neuronal injury marker activating transcription factor-3 in IB4+ and NF200+ sensory neurons as well as an increase in the chemokines CCL2 and CCL3 in the lumbar dorsal root ganglion. In the central nervous system, PTX induced significant astrocyte activation in the spinal cord dorsal horn, and both PTX and OXA caused reduction of P2ry12+ homeostatic microglia, with no measurable changes in IBA-1+ microglia/macrophages in the dorsal and ventral horns. We also found that PTX induced up-regulation of several inflammatory cytokines and chemokines (TNF-α, IFN-γ, CCL11, CCL4, CCL3, IL-12p70 and GM-CSF) in the spinal cord. Overall, these findings suggest that PTX and OXA cause distinct pathological changes in the periphery and nervous system, which may contribute to chemotherapy-induced neuropathic pain

Oxaliplatin-induced haematological toxicity and splenomegaly in mice.

PurposeHaematological toxicities occur in patients receiving oxaliplatin. Mild anaemia (grade 1-2) is a common side effect and approximately 90% of recipients develop measurable spleen enlargement. Although generally asymptomatic, oxaliplatin-induced splenomegaly is independently associated with complications following liver resection for colorectal liver metastasis and separately with poorer patient outcomes. Here, we investigated oxaliplatin-induced haematological toxicities and splenomegaly in mice treated with escalating dosages comparable to those prescribed to colorectal cancer patients.MethodsBlood was analysed, and smears assessed using Wright-Giemsa staining. Paw coloration was quantified as a marker of anaemia. Spleen weight and morphology were assessed for abnormalities relating to splenomegaly and a flow cytometry and multiplex cytokine array assessment was performed on splenocytes. The liver was assessed for sinusoidal obstructive syndrome.ResultsBlood analysis showed dose dependent decreases in white and red blood cell counts, and significant changes in haematological indices. Front and hind paws exhibited dose dependent and dramatic discoloration indicative of anaemia. Spleen weight was significantly increased indicating splenomegaly, and red pulp tissue exhibited substantial dysplasia. Cytokines and chemokines within the spleen were significantly affected with temporal upregulation of IL-6, IL-1α and G-CSF and downregulation of IL-1β, IL-12p40, MIP-1β, IL-2 and RANTES. Flow cytometric analysis demonstrated alterations in splenocyte populations, including a significant reduction in CD45+ cells. Histological staining of the liver showed no evidence of sinusoidal obstructive syndrome but there were signs suggestive of extramedullary haematopoiesis.ConclusionChronic oxaliplatin treatment dose dependently induced haematological toxicity and splenomegaly characterised by numerous physiological and morphological changes, which occurred independently of sinusoidal obstructive syndrome

Chemotherapy-induced astrocyte activation in the spinal cord dorsal horn.

<p>GFAP immunohistochemistry in L3-L5 spinal cord was carried out on day 13 post 1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control). Column graph showing the percentage of GFAP immunoreactivity in spinal cord dorsal and ventral horns, and representative images depicting GFAP immunoreactivity in the dorsal horn. GFAP immunoreactivity was significantly higher in the dorsal horn of PTX-treated mice compared with saline controls (**P<0.01), with no significant changes in the ventral horn. Scale bar = 50μm. n = 4, one-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Effect of chemotherapy on mechanical sensitivity in C56BL/6J male mice.

<p>Time course of mechanical paw withdrawal threshold (PWT; in grams) of male C56BL/6J mice treated with paclitaxel (PTX), oxaliplatin (OXA) or saline (control). Measurements were taken from baseline before chemotherapy until day 27, with chemotherapy injections administered at days 0, 2, 4 and 6. Compared to baseline thresholds, significant decrease in PWT was seen on days 8, 13, and 16 in the PTX-treated group, and on days 8, 13, 16 and 27 in the OXA-treated group. **(P<0.01); ***(P<0.001); ****(P<0.0001) indicate significant difference between baseline and post-chemotherapy time points in the PTX group. ⁺⁺(P<0.01); ⁺⁺⁺(P<0.001); ⁺⁺⁺⁺(P<0.0001) indicate significant difference between baseline and post-chemotherapy time points in the OXA group. PTX (n = 16), OXA (n = 16), Saline (n = 15). RM two-way ANOVA followed by Bonferroni’s multiple comparisons test. Data are expressed as mean±SEM.</p

Chemotherapy-induced neuronal injury in the lumbar DRG.

<p>ATF-3, IB4, CGRP and NF200 immunohistochemistry in L3-L4 DRG was carried out on day 13 post-1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control). (A) Column graph and representative images depicting neuronal ATF-3 expression in the DRG, which was significantly higher in the PTX group compared to saline control group (*P<0.05). Scale bar = 50 μm. (B) Column graph and representative images showing double immunolabelling of ATF-3 with IB4, CGRP and NF200 in the DRG of PTX-treated mice. ATF-3 was predominantly expressed in IB4+ and NF200+ DRG neurons in the PTX group (*P<0.05). Scale bar = 50μm. n = 3–4, one-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Changes in regulatory T-cell population in inguinal lymph nodes following chemotherapy.

<p>Flow cytometry of lymphocytes to characterise regulatory T-(T-reg)-cell changes in inguinal lymph nodes on days 7 and 13 post-1^st injection of paclitaxel (PTX), oxaliplatin (OXA) or saline (control) was carried out. CD4+ cells were first gated from lymphocyte singlets (A), followed by consecutive gating of CD25+FoxP3+ cells (B). Column graphs of CD4+CD25+FoxP3+ T-reg cell populations in the lymph nodes on day 7 (C) and day 13 (D) are expressed as a percentage of lymphocyte singlets. A significant decrease in T-reg cells was seen in OXA-treated mice on both day 7 (n = 6, ****P<0.0001) and day 13 (n = 4, *P<0.05) compared to saline control mice. One-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p

Cytokine and chemokine changes in the DRGs following chemotherapy.

<p>Cytokine and chemokine changes in the DRGs following chemotherapy.</p

Cytokine and chemokine changes in the spinal cord following chemotherapy.

<p>Cytokine and chemokine changes in the spinal cord following chemotherapy.</p

Systemic inflammatory changes in the blood and spleen.

<p>Flow cytometry of lymphocytes to characterise inflammatory changes in the blood and spleen was carried out on day 7 post-1^st paclitaxel (PTX), oxaliplatin (OXA) or saline (control) injection. Mononuclear cells were first gated (A), followed by consecutive gating of singlets (B). For blood, singlets were further gated for CD45+ lymphocytes (C). Representative flow cytometric plots and column graphs of CD45+CD4+ (D) and CD45+CD8+ (E) T-cell populations in the blood (expressed as a percentage of CD45+ lymphocyte singlets), and total IL-4+ lymphocytes (F) in the spleen (expressed as a percentage of lymphocyte singlets). Significant increase in CD45+CD4+ and CD45+CD8+ T-cell populations in the blood of PTX- and OXA- treated mice (n = 10), and IL-4 positive lymphocytes in the spleens of OXA-treated mice (n = 7–9) compared to saline controls were found. *P<0.05; **P<0.01; ***P<0.001 indicate significant difference between PTX/OXA and saline controls. One-way ANOVA followed by Bonferroni's multiple comparison’s test. Data are expressed as mean±SEM.</p