Sensitization of human and rat nociceptors by low dose morphine is toll-like receptor 4-dependent

Culturing rat DRG neurons

Primary cultures of dorsal root ganglia (DRG) were made from 220 to 235 g adult male Sprague-Dawley rats, as described previously.^21–24 Under isoflurane anesthesia, rats were decapitated, and the dorsum of their vertebral column surgically removed; L₄ and L₅ DRGs were rapidly extracted, bilaterally, chilled and desheathed in Hanks’ balanced salt solution (HBSS), on ice. DRG were treated with 0.25% collagenase Type 4 (Worthington Biochemical Corporation, Lakewood, NJ, USA) in HBSS for 18 min at 37°C, and then with 0.25% trypsin (Worthington Biochemical Corporation) in calcium- and magnesium-free PBS (Invitrogen Life Technologies, Grand Island, NY USA) for 6 min, followed by three washes and trituration in Neurobasal-A medium (Invitrogen Life Technologies) to produce a single-cell suspension. This suspension was centrifuged at 1000 r/min for 3 min followed by re-suspension in Neurobasal-A medium that was supplemented with 50 ng/mL nerve growth factor, 100 U/mL penicillin/streptomycin, B-27, GlutaMAX and 10% FBS (Invitrogen Life Technologies). Cells were then plated on cover slips and incubated at 37°C in 3.5% CO₂ for at least 24 h before they were used in electrophysiology experiments.

Culturing human DRG neurons

Human DRG neuronal culture from a single male donor were purchased from AnaBios (San Diego, CA, USA). Dissociated human DRG neuronal suspension in a protective solution from this donor was shipped to UCSF on ice. They were delivered within hours after preparation of the suspension from donor tissue at the AnaBios facility. All samples underwent visual inspection for tissue integrity in our laboratory and in parallel at AnaBios, using fluorescent microscopy and electrophysiology. Human tissue originating from AnaBios was obtained legally with consent from US-based organ donors, adhering to United Network for Organ Sharing (UNOS) donor screening and consent standards. AnaBios follows procedures endorsed by the US Centers for Disease Control (CDC), subject to biannual inspections by the US Department of Health and Human Services (DHHS). Distribution of tissue to investigators by AnaBios is governed by internal IRB procedures and is in compliance with Health Insurance Portability and Accountability Act (HIPAA) regulations. All organ transfers to AnaBios are traceable and periodically reviewed by US Federal authorities, ensuring the privacy of donor information during studies involving human DRGs.

Pursuant to an AnaBios protocol, upon arrival at UCSF (San Francisco, CA, USA) the content of the transportation vial (2 mL of protective media, containing ∼1000 neurons) was transferred to 9 mL of wash media (DMEM/F12 with penicillin/streptomycin [Lonza; Allendale, NJ]), then recovered by centrifugation (∼350 xG) at room temperature for 3 min, and resuspended in 0.6 mL of culturing media provided by AnaBios, made on the basis of DMEM/F12 with penicillin/streptomycin and supplements: 10% horse serum (Thermo Fisher Scientific, Rockford, IL, USA), 2 mM glutamine, 25 ng/mL hNGF (Cell Signaling Technology, Danvers, MA, USA) and 25 ng/mL GDNF (PeproTech, Rocky Hill, NJ, USA). ²⁵

Cells from dissociated DRG were plated on 3 round (15 mm diameter) glass coverslips (0.2 mL of suspension per coverslip, in separate Petri dishes) that had been precoated with poly-D-lysine (reported by AnaBios to be important for survival) (Neuvitro Corp., Camas, WA, USA). One hour after plating, 1 mL of the culture media was gently added and coverslips fractured such that 6-8 individual pieces of a coverslip that could be used for electrophysiology recordings was obtained. As recommended by AnaBios, human DRG neurons were used after incubating for 48 h (5% CO2, 37°C), for up to day 10. Culture media in Petri dishes was exchanged every 2 days by replacing half of the media.

Whole-cell patch-clamp electrophysiology

Following placement of individual coverslip fragments, plated with cells from dissociated human or rat DRGs, in the recording chamber, culture medium was substituted with the solution used to perform electrophysiology, Tyrode’s solution containing 140 mm NaCl, 4 mm KCl, 2 mm MgCl₂, 2 mm CaCl₂, 10 mm glucose, and 10 mm HEPES, adjusted to pH 7.4 with NaOH, with an osmolarity of 310 mOsm/kg. The recording chamber had a volume of 150 µl, and its perfusion system had a flow rate of 0.5–1 ml/min. Electrophysiology experiments were conducted at room temperature (20–23°C).

Cells were identified as neurons by their double birefringent plasma membranes.^26,27 Whole-cell patch-clamp recordings, performed in current clamp mode, were used to evaluate for changes in the excitability of cultured rat and human DRG neurons. Holding current was adjusted to maintain membrane potential at −70 mV. Rheobase, defined as the minimum magnitude of a current step needed to elicit an action potential (AP), was determined through a testing protocol utilizing a series of square wave pulses with current magnitude increasing by a constant step every sweep, until an AP was elicited. An initial estimate of rheobase was made with 500-pA increments (0.5 – 4 nA). The increments were then adjusted to achieve 5%–10% precision of the rheobase estimate.^24,28

Recording electrodes were fashioned from borosilicate glass capillaries (0.84/1.5 mm i.d./o.d., Warner Instruments, LLC) using a Flaming/Brown P-87 microelectrode puller (Sutter Instrument Co, Novato, CA, USA). After being filled with a solution containing 130 mm KCl, 10 mm HEPES, 10 mm EGTA, 1 mm CaCl₂, 5 mm MgATP, and 1 mm Na-GTP; pH 7.2 (adjusted with Tris-base), resulting in 300 mOsmol/kg osmolarity, the recording electrode resistance was approximately 2 MΩ. Junction potential was not adjusted, and series resistance was below 10 MΩ at the end of recordings, without compensation. Recordings were conducted using an Axon MultiClamp 700 B amplifier, filtered at 20 kHz, and sampled at 50 kHz through an Axon Digidata 1550B controlled by pCLAMP 11 software (all from Molecular Devices LLC, San Jose, CA, USA).

To ensure the stability of baseline current, drugs were applied at least 5 min after the establishment of whole-cell configuration.

Drugs and media

The following drugs were used in this study: Morphine sulfate salt pentahydrate, NaCl, KCl, MgCl₂, CaCl₂, NaOH, MgATP, Na-GTP, D-Glucose, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma Aldrich, St Louis, MO, USA), LPS-RS Ultrapure (a selective TLR4 antagonist, lipopolysaccharide from Rhodobacter 9 sphaeroides, purchased from InvivoGen, San Diego, CA, USA), Collagenase Type 4, Trypsin (Worthington Biochemical Corporation, Lakewood, NJ, USA), Calcium- and Magnesium-free HBSS, Calcium- and Magnesium-free PBS, Neurobasal-A medium, B-27 supplement, GlutaMAX, Fetal Bovine Serum (FBS) (Invitrogen Life Technologies, Grand Island, NY USA), rat (recombinant) nerve growth factor (NGF)-beta, glutamine (Sigma Aldrich), penicillin/streptomycin, DMEM/F12 with penicillin/streptomycin (Lonza, Allendale, NJ, USA), horse serum (Thermo Fisher Scientific, Rockford, IL, USA), human NGF (hNGF, Cell Signaling Technology, Danvers, MA, USA), human GDNF (Peprotech, Rocky Hill, NJ, USA).

Stock solution of LPS-RS Ultrapure was prepared in purified water (1 mg/mL) and stored in 50 µL vials at −20°C. One vial was thawed on the day of the experiment and stored at 4°C. Final concentration of LPS-RS Ultrapure (10 µg/mL) was achieved by 1:99 dilution in Tyrode’s solution, performed just before it was used in experiments.

Stock solution of morphine sulfate (0.5 mM) was prepared freshly from powder (0.76 mg/mL, in purified water) on the day of the experiment and stored at 4°C. The final concentration of morphine in perfusion solution was selected to be 100 nM (76 ng/mL; 1:4999 dilution of the stock solution, performed in two steps, 1:99 in purified water then 1:49 in Tyrode’s solution); we consider a low concentration, as it is 10-100x lower than then concentration used in most in vitro studies (1-10 µM).^29–32 We estimate that plasma concentration of 100 nM would be produced by a single intravenous sub-analgesic dose (0.15 mg/kg) based on the reported findings that, on average, a dose of 3.0 mg of morphine resulted in plasma level of 40.2 ng/mL in human subjects who had an average weight of 56.3 kg (Table 1 and 2 from ¹⁹ ). We have previously shown that, when performing a dose response curve, in the rat, morphine induces analgesia at doses starting at ∼1 mg/kg. ¹⁴

Statistical analysis

Rheobase was measured before and 5 min after application of morphine. Magnitude of the effect of morphine was expressed as percentage reduction in rheobase (i.e., value before morphine administration was subtracted from value after, then the difference was divided by pre drug baseline). The following statistical tests were used: one-sample two-tailed Student's t-test versus zero and two-sample unpaired two-tailed Student's t-test.

Prism 10.1 (GraphPad Software) was used to generate graphics and to perform statistical analyses; p < .05 is considered statistically significant. Data are presented as mean ± SEM.

Nociceptor sensitization by low dose morphine

To test the hypothesis that low dose morphine sensitizes small-diameter human and rat DRG neurons, we performed in vitro patch-clamp electrophysiology, evaluating the effect of our low concentration of morphine (100 nM, i.e. 76 ng/mL, corresponding to a single sub-analgesic dose of 0.15 mg/kg)^14,19 on neuronal excitability. Rheobase, the minimal sustained current required to generate an action potential (AP), was selected for our measure of neuronal excitability as it is a well-defined electrophysiological property that reflects excitability in DRG neurons.^{25,28,33–35} Percentage decrease in rheobase was used as the measure of nociceptor sensitization. The effect of 100 nM morphine on rheobase was evaluated in putative C-type nociceptive (small-diameter) neurons from rat (soma diameter <30 µm)^36–39 and human (soma diameter 40-60 µm) DRG.^40–42 We found that low dose morphine induces a significant decrease in rheobase in small-diameter rat (26 ± 7%, n = 6, *p = .013, t₍₅₎ = 3.8, Figure 1(b); illustrative traces in Figure 1(a)) and human (36 ± 7%, n = 9, **p = .0014, t₍₈₎ = 4.8, Figure 1(d); illustrative traces in Figure 1(c)) DRG neurons.

Prevention of nociceptor sensitization by low dose morphine with a selective TLR4 antagonist

We next evaluated the contribution of TLR4 to low dose morphine-induced sensitization of rat and human small-diameter DRG neurons, in vitro. In both rat and human small-diameter DRG neurons that were pretreated with the selective TLR4 antagonist, LPS-RS Ultrapure (10 μg/mL, for 30 min before, during, and continued after the exposure to morphine), reduction in rheobase induced by low dose morphine (relative to baseline in the same cell) was significantly attenuated, compared to morphine treatment without the TLR4 antagonist (rat: ^#p = .018, t₍₉₎ = 2.9; human: ^& p = .031, t₍₁₃₎ = 2.4) and was not significantly different from zero (no effect) (rat: 0 ± 5%, n = 5, 95% CI = (−14 .. +15)%, p = .96, t₍₄₎ = 0.05, Figure 1(b); illustrative traces in Figure 1(a); human: 9 ± 7%, n = 6, 95% CI = (−10 .. +28)%, p = .29, t₍₅₎ = 1.2, Figure 1(c); illustrative traces in Figure 1(d)).