Intrathecal administration of conditioned serum from different species resolves Chemotherapy-Induced neuropathic pain in mice via secretory exosomes

Abstract

Chemotherapy-induced peripheral neuropathy (CIPN) is the most prevalent neurological complication of chemotherapy for cancer, and has limited effective treatment options. Autologous conditioned serum (ACS) is an effective biologic therapy used by intra-articular injection for patients with osteoarthritis. However, ACS has not been systematically tested in the treatment of peripheral neuropathies such as CIPN. It has been generally assumed that the analgesic effect of this biologic therapy results from augmented concentrations of anti-inflammatory cytokines and growth factors. Here we report that a single intrathecal injection of human conditioned serum (hCS) produced long-lasting inhibition of paclitaxel chemotherapy-induced neuropathic pain (mechanical allodynia) in mice, without causing motor impairment. Strikingly, the analgesic effect of hCS in our experiments was maintained even 8 weeks after the treatment, compared with non-conditioned human serum (hNCS). Furthermore, the hCS transfer-induced pain relief in mice was fully recapitulated by rat or mouse CS transfer to mice of both sexes, indicating cross-species and cross-sex effectiveness. Mechanistically, CS treatment blocked the chemotherapy-induced glial reaction in the spinal cord and improved nerve conduction. Compared to NCS, CS contained significantly higher concentrations of anti-inflammatory and pro-resolving mediators, including IL-1Ra, TIMP-1, TGF-β1, and resolvins D1/D2. Intrathecal injection of anti-TGF-β1 and anti-Il-1Ra antibody transiently reversed the analgesic action of CS. Nanoparticle tracking analysis revealed that rat conditioned serum contained a significantly greater number of exosomes than NCS. Importantly, the removal of exosomes by high-speed centrifugation largely diminished the CS-produced pain relief, suggesting a critical involvement of small vesicles (exosomes) in the beneficial effects of CS. Together, our findings demonstrate that intrathecal CS produces a remarkable resolution of neuropathic pain mediated through a combination of small vesicles/exosomes and neuroimmune/neuroglial modulation.

Keywords: ACS (autologous conditioned serum); CIPN (chemotherapy-induced peripheral neuropathy); Exosomes, glial cells; IL-1Ra (interleukin-1 receptor antagonist); Neuroinflammation; Spinal cord; conditioned serum (CS); non-conditioned serum (NCS).

Figure 1.

(A) Schematic of blood collection and blood incubation in human, rat, and mouse samples. (B) Schematic of serum correction for conditioned serum (CS) and non-conditioned serum (NCS) from humans (hCS and hNCS), rats (rCS and rNCS), and mice (mCS and mNCS). (C) Schematic of a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) by four injections of paclitaxel on day 1, 3, 5, and 7 in CD1 mice. (C) Schematic of behavioral tests for mechanical pain (von Frey tests) and cold pain (acetone test). (D) Schematic of a single intrathecal injection of NCS and CS (from human, mouse, and rat) to CD1 mice of both sexes.

Figure 2.

Intrathecal injection of human CS (hCS, from a male doner) alleviates neuropathic pain symptom following PTX-induced CIPN in male mice. (A, B) Von Frey tests for mechanical pain showing paw withdrawal threshold (PWT, A) and paw withdrawal frequency (PWF, B). (C) Acetone test showing cold pain. (D) Open-field test in hCS and hNCS treated animals. hCS produces long-term reduction of PTX-induced mechanical allodynia by increasing PWT (A) and decreasing PWF (B), without effects on cold pain (C) and motor function (D). n=5 male mice per group. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA, followed by Bonferroni posthoc test. hCS, human conditioned serum; hNCS, human non-conditioned serum.

Figure 3.

Intrathecal human CS (hCS, from a male donor) treatment significantly improves nerve conduction velocity in male and female mice with CIPN. (A) Schematic of sensory conductance measurement. Two stimulation electrodes were inserted into a hind paw with 1 cm between them and the recording cuff electrode was inserted to the sciatic nerve (1 ms stimulation 10V). (B) Representative nerve conductance trace. left: control mice. right: hCS-treated mice. (C,D) Quantification of nerve conductance velocity (C) and amplitude (D). n = 7 mice for naïve control, n = 5 mice for hNCS with PTX, n = 7 mice for hCS with PTX. *p<0.05, Unpaired t-test.

Figure 4.

Intrathecal injection of rat CS (rCS) alleviates neuropathic pain symptom in mice. (A, B) Von Frey tests for mechanical pain showing PWT (A) and PWF (B). Note that rCS with 24h incubation, but not 12h incubation, produces long-term reduction of PTX-induced mechanical allodynia by increasing threshold (A) and decreasing frequency (B). n=5 male mice per group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA, followed by Bonferroni posthoc test. Male donors and receivers were tested.

Figure 5.

Intrathecal rat ACS (rCS) treatment inhibits glial reaction in the spinal cord dorsal horn of male mice with CIPN. (A,B) Double immunofluorescence staining of the lumbar spinal dorsal horn sections showing GFAP and IBA1 immunostaining following rNCS (A) and rCS treatment (B) in PTX mice. (C-D) Quantification of the integrated density of GFAP (C) and IBA1 (D) in the lumbar spinal dorsal horn of PTX mice treatment with rNCS and rCS (n = 5/group). *p< 0.05, **p< 0.01 vs. control serum (rNCS). Unpaired two-tailed Student’s t test. Spinal cord tissues were harvested one week after the rCS treatment. Male donors and receivers were tested.

Figure 6.

ELISA analyses of human and rat CS (hCS, rCS) and NCS (hNCS, rNCS). (A-F) Human concentrations of cytokines and resolvins in hCS and control serum (hNCS). (A) IL-1Ra levels in hCS and hNCS. (B) TIMP-1 levels in hCS and hNCS. (C,E) Standard curves of RvD1 and RvD2 ELISA. (D,F) RvD1 and RvD2 levels in hCS and hNCS. *p<0.05, ***p<0.001; paired student’s t-est. n=3 samples from two healthy male volunteers (blue) and one healthy female donor (red). (G, H) Rat serum concentrations of TGF-β1 (G) and TIMP-1 (H) in rCS and control serum (rNCS). *p<0.05, ****p<0.0001; n=8 (rNCS), n=8 (rCS), unpaired student’s t-test.

Figure 7.

Anti-TGF-β1 and anti-IL1ra neutralizing antibodies transiently reverses the anti-allodynic effects of rCS in mice. (A, B) Von Frey tests for PWT (A) and PWF (B) showing a temporary reversal of hCS-induced analgesia at 1 hour post-injection with 4 μg of anti-TGF-β1 antibody. (C, D) Von Frey tests for PWT (C) and PWF (D) showing a temporary reversal of rCS-induced analgesia at 1-3 h post-injection with 4 μg of anti-IL-1ra antibody. Intrathecally-injected IgG was included as control. *p<0.05, **p<0.01, ***, p<0.001, n=5 per group, two-way ANOVA, followed by Bonferroni posthoc test. Male donors and receivers were tested.

Figure 8.

Nanoparticle tracking analysis showing increased exosome concentration in male rat CS (rCS). (A) Paradigm of nanoparticle tracking analysis. (B) Size distribution and concentrations of extracellular vesicles in rNCS, rCS, and U-rCS in each of five rats and all rats combined. (C) Particle density per mL serum. (D) Normalized extracellular vesicle (EV) concentrations as percentage of rNCS. **p<0.01, ***p<0.001, n=5 rats, one-way ANOVA, followed by Bonferroni posthoc test. rUCS, rat CS after ultracentrifugation. Note that EV is deleted in rUCS.

Figure 9.

Effects of ultracentrifugation on rCS-induced analgesia in CIPN mice. (A) Schematic of ultracentrifugation (100,000 x g, 120 min, 4°C). (B, C) Von Frey tests for paw withdrawal threshold (B) and withdrawal frequency (C). Ultracentrifuged rCS (U-rCS) showed significantly decreased analgesic effects versus ACS for all measured timepoints up to 28 days following intrathecal injection in CIPN mice. Intrathecally injected ultracentrifuged rNCS (U-rNCS) was used as control. *p<0.05, **p<0.01, ****p<0.001, **p<0.0001, n=5 per group, two-way ANOVA, followed by Bonferroni posthoc test. Male donors and receivers were tested.