Animals Models:
Adult male and female 8-weeks old C57BL/6J mice (Jackson Lab, Bar Harbor, ME) were used in this study. Mice were acclimatized at least 7 days before use and maintained throughout in standard housing conditions with access to food and water (24 ± 2°C temperature; 50 ± 10% relative humidity). Mice receiving morphine were injected with a ramping dose for four days starting at day one with 20mg/kg i.p b.i.d, followed by two days of 40mg/g i.p b.i.d, and a final day of 80mg/kg i.p b.i.d morphine. Sodium butyrate (25mM-800 mM wt/v) was given by oral gavage twice daily for 4 days starting on the first day of morphine i.p injection. For the studies on paclitaxel induced hypersensitivity, adult male mice received 8 mg/kg, i.p.of paclitaxel or 1:1:18 vehicle (1 volume cremophor EL, 1 volume ethanol, 18 volume saline) i.p. injections on alternating days (days 1, 3, 5, 7) to a cumulative dose of 32 mg/kg as described previously53. Paclitaxel cohorts receiving the butyrate treatment were treated twice daily with a 250mM oral gavage of sodium butyrate for 14 days starting on the first day of paclitaxel i.p injection. Mice in the naloxone cohort were injected with a ramping dose for four days starting at day one with 2mg/kg i.p b.i.d, followed by two days of 4mg/g i.p b.i.d, and a final day of 8mg/kg i.p b.i.d naloxone. Animals were excluded from the study if they lost more than 20% of body weight, or developed an adverse reaction to injections over the course of treatment period. Investigators were blinded to the animal’s treatment condition during data collection for behavioral experiments.
Ethics Declarations:
All procedures and methodologies were conducted in accordance with the procedures reviewed and approved by the Institutional Animal Care and Use committee at Virginia Commonwealth University (VCU IACUC). All methods are reported in accordance with ARRIVE guidelines54 .
Hot plate thermal hyperalgesia assay
Animals receiving morphine were tested on day 5, 18 hours after the last injection of morphine for the development of thermal hyperalgesia by utilizing a hot plate assay. The hot plate was set to 50 and animals were placed in the center of the heated area and allowed to engage in the assay for 30s. During the assay if the animals engaged in hind paw licking, hind paw shaking, or jumping behaviors the assay was ended and the total time spent on the hot plate was recorded.
Acetone evaporation assay: Animals received 8mg/kg paclitaxel or vehicle (1:1:18, cremophor, ethanol, saline) every other day for 7 days, and were subsequently tested for the development of cold allodynia by acetone evaporation. Acetone evaporation produces a non-noxious sensation of coolness approximately equivalent to 15–21°C when applied to the skin55. Animals were tested at Day 7 and Day 14 post-paclitaxel treatment using a clear Plexiglas box with wire bottom to allow for access to the plantar surface of the animal’s hind paw. Animals were acclimated for approximately 1 hour prior to testing after which time acetone was applied via a gavage needle to produce a drop (approximately 100µl) onto the plantar surface of the left or right paw. Animal behavior was recorded for 30s and the behavioral response graded on a scale of 0–3 in increasing intensity for both paws (i.e 0, no response; 1, brisk withdrawal or flick of the paw; 2, licking of the paw; 3, prolonged licking/biting of the hind paw)55. Animals are then left undisturbed for 10 minutes and the trial repeated on the other paw. Scores were averaged across all trials to produce a single allodynia score. For comparison, 30°C water was used as a control and applied to the paw and behaviors scored as above with acetone. the cumulative time spent engaging (flicking, shaking, grooming, licking etc.) the stimulated paw after acetone application was also quantified.
DRG Isolation
Isolation of DRG neurons was carried out as described in previous studies13,32. Following sacrifice by cervical dislocation, DRG were harvested from spinal levels L5-S1 (those supplying the lower gastrointestinal tract) and immediately placed in cold (4°C) Hanks’ Balanced Salt Solution (HBSS). Ganglia were then incubated (37°C) for 18 min in HBSS with 36.6 ug/mL papain, washed with HBSS, and incubated for 1hr in HBSS with 1.5 mg/mL collagenase. Tissues were gently triturated and centrifuged for 5 min at 1,000 rpm. The colonic media is decanted, and cells resuspended in neurobasal A medium containing 1% FBS, 1x B-27 supplement, 2 mM L-glutamine, 10 ng/mL GDNF, and penicillin/streptomycin/amphotericin B. The suspension was plated on poly-D-lysine/laminin coated coverslips and incubated (37°C) for at least 24 hr.
Electrophysiological Recordings and Solutions: Coverslips were transferred to a microscope stage plate and continuously perfused with external physiologic saline solution (ePSS). Standard ePSS contained the following in mM: 135 NaCl, 5.4 KCl, 0.33 NaH2PO4, 5 HEPES, 1 MgCl2, 2 CaCl2, and 5 glucose (pH adjusted to 7.4 with 1 M NaOH). DRG nociceptors of the small, C and Aδ fiber types56, low capacitance (< 30pF) neurons were selected, and a GΩ seal achieved via pulled and fire-polished (2.5–4.5 MΩ) borosilicate glass capillaries. Standard internal PSS (iPSS) contains, in mM: 100 L-aspartic acid (K salt), 30 KCl, 4.5 Na2ATP, 1 MgCl2, 10 HEPES, 6 EGTA, and 0.5 NaGTP (pH adjusted to 7.2 with 3 M KOH). Recordings were made using a Axopatch 200B amplifier and Clampex/Clampfit 11.0 data acquisition software was used for analyses. Action potential (AP) threshold and rheobase estimates were measured from resting current, a 10pA stimulus pulse is applied in 10pA steps starting from − 30pA for 10ms. For assessing multiple action potential firing events, a 500ms (150ms for paclitaxel) pulse period was used. Phase plots were generated by plotting the first derivative of the AP against membrane potential, maximum rate changes between successive potentials were measured during the rising phase of the AP
Colon tissue conditioned media collection
In order to demonstrate whether inflammatory mediators released within the colon enhance neuronal excitability, we tested the effect of colon conditioned media (CCM) on DRG neuronal excitability, similar to previous studies13. Following sacrifice by cervical dislocation, full circumference colon segments 5 mm in length are resected and placed in 250 µL of neurobasal A medium containing 1% FBS, 1x B-27 supplement, 2 mM L-glutamine, 10 ng/mL GDNF, and penicillin/streptomycin/amphotericin B. The samples were incubated (37°C) for 24h and then the colon conditioned media was collected and either frozen in liquid nitrogen or transferred to freshly isolated naïve DRG neuron cultures. An additional 200 µL of fresh medium is added to the culture, which is then incubated (37°C) for 24 h before performing electrophysiology experiments.
Data analysis. Sample sizes were determined using G*Power to detect 80% power for a medium effect size (Cohen’s d = 0.5) and an alpha level of 0.05. Data were analyzed using Graphpad Prism 9 (La Jolla, CA). Data were analyzed via a two-way ANOVA that included relevant drug treatment, and butyrate treatment as independent variables. Bonferroni’s post hoc test was used for those analyses which indicated a significant interaction (p < 0.05). A three-way ANOVA with Tukey’s post-hoc test was utilized when analyzing excitability data comparing increasing levels of stimulation against relevant drug treatment and butyrate treatment respectively on the number of evoked potentials.