Spinal nociceptive transmission by mechanical stimulation of bone marrow

Abstract

Background: Since bone marrow receives innervation from A-delta and C-fibers and since an increase in intramedullary pressure in bone marrow may induce acute pain in orthopedic patients during surgery and chronic pain in patients with bone marrow edema, skeletal pain may partly originate from bone marrow. Intraosseous lesions, such as osteomyelitis and bone cancer, are also known to produce cutaneous hypersensitivity, which might be referred pain from bone. However, little is known about pain perception in bone marrow and referred pain induced by bone disease. Thus, we carried out an in vivo electrophysiological study and behavioral study to determine whether increased intraosseous pressure of the femur induces acute pain and whether increased intraosseous pressure induces referred pain in the corresponding receptive fields of the skin.

Results: Intraosseous balloon inflation caused spontaneous pain-related behavior and mechanical hyperalgesia and allodynia in the lumbosacral region. Single neuronal activities of spinal dorsal horn neurons were extracellularly isolated, and then evoked responses to non-noxious and noxious cutaneous stimuli and intraosseous balloon inflation were recorded. Ninety-four spinal dorsal horn neurons, which had somatic receptive fields at the lower back and thigh, were obtained. Sixty-two percent of the wide-dynamic-range neurons (24/39) and 86% of the high-threshold neurons (12/14) responded to intraosseous balloon inflation, while none of the low-threshold neurons (0/41) responded to intraosseous balloon inflation. Spinally administered morphine (1 µg) abolished balloon inflation-induced spontaneous pain-related behavior and mechanical hyperalgesia in awake rats and also suppressed evoked activities of wide-dynamic-range neurons to noxious cutaneous stimulation and intraosseous balloon inflation.

Conclusions: The results suggest that mechanical stimulation to bone marrow produces nociception, concomitantly producing its referred pain in the corresponding skin fields. These mechanisms might contribute to pain caused by skeletal diseases.

Keywords: Skeletal pain; in vivo electrophysiology; referred pain.

Figure 1.

Intraosseous and cutaneous sensory convergence at dorsal root ganglion neurons. Labeled neurons in the L3 dorsal root ganglion (DRG) after application of Fluoro-Gold (FG) to the medullary cavity of the femur and application of cholera toxin subunit B (CTB) to the skin of the lumbosacral region. The arrowheads indicate a neuron double-labeled with FG and CTB. Scale bar, 100 µm.

Figure 2.

Chronic implantation of a balloon catheter in the femur. A skin incision was made on the medial side of the right knee, and the proximal patellar ligament of the femur was severed, revealing the synovial space of the knee joint. A 22-gauge needle was used to core between the femoral condyles and into the medullary cavity of the right femur. A diamond drill burr, 1 mm in diameter, was used to prime the opening of the hole made by the 22-gauge needle. A balloon catheter used for coronary angioplasty in patients with coronary artery disease (PTCA catheter, RX-2, 2 × 15 mm, TERUMO Co., Tokyo, Japan) was implanted in the medullary cavity of the femur for increasing intraosseous pressure of the femur. The balloon catheter was then tunneled subcutaneously to emerge at the neck. To stimulate intraosseous receptors, the balloon was inflated with saline using an inflation device (Encore™ 26, Boston Scientific Japan, Tokyo, Japan).

Figure 3.

Relationship between spontaneous pain-related behavior and intraosseous pressure of the femur. Guarding behavior and flinches induced by balloon inflation in the medullary cavity of the femur at pressure from 50 to 400 kPa. Guarding behavior (a) and flinches (b) increased in accordance with an increase in balloon inflation pressure. Data are expressed as means ± SEMs.

Figure 4.

Effects of intrathecal morphine and naloxone on spontaneous pain-related behavior induced by intraosseous balloon inflation. Effects of intrathecal (i.t.) morphine and i.t. naloxone on spontaneous pain-related behavior assessed as the time spent guarding (a) and the number of flinches (b) induced by balloon inflation in the medullary cavity of the femur (200 kPa). Guarding behavior and flinches without intraosseous stimulation were recorded before injection (control), 15 min after i.t. injection of morphine (morphine, 1 µg), and 15 min after injection of naloxone (naloxone, 10 µg). After recording of pain-related behavior without intraosseous stimulation, guarding behavior and flinches induced by balloon inflation in the medullary cavity of the femur were recorded. I.t. administration of morphine suppressed intraosseous stimulation-induced guarding behavior and flinches. I.t. naloxone antagonized the effect of morphine. Data are expressed as means ± SEMs (n = 7). *p < 0.01 versus control without intraosseous stimulation, #p < 0.01 versus naloxone without intraosseous stimulation.

Figure 5.

Effects of intrathecal morphine and naloxone on mechanical hyperalgesia induced by intraosseous balloon inflation. Effects of i.t. morphine and i.t. naloxone on mechanical withdrawal threshold in response to punctate noxious stimuli (a) and dynamic non-noxious stimuli (b) with sustained balloon inflation (200 kPa) in the medullary cavity of the femur. After recording withdrawal responses without intraosseous stimulation, withdrawal responses with intraosseous stimulation were recorded. Then, withdrawal thresholds with intraosseous stimulation (200 kPa) were recorded 15 min after i.t. morphine (morphine) and 15 min after i.t. naloxone (naloxone). Intraosseous stimulation decreased the withdrawal threshold and increased the withdrawal response frequency in the control rats. I.t. administration of morphine reduced intraosseous stimulation-induced mechanical hyperalgesia and allodynia. I.t. naloxone antagonized the effect of morphine on the withdrawal thresholds induced by balloon inflation. Data are expressed as medians (horizontal line) and boxes and whiskers with first and third quartiles (box) and as minimum and maximum (whiskers) or means ± SEMs (n = 7). *p < 0.05 versus control without balloon inflation; p < 0.05 versus naloxone without balloon inflation.

Figure 6.

Recording sites of neurons. Recording sites of neurons that responded to intraosseous stimulation (a) and neurons that did not responded to intraosseous stimulation (b). Black filled circles, wide-dynamic-range (WDR) neurons that responded to inflation of the balloon in the medullary cavity of the femur; black open circles, WDR neurons that did not respond to inflation of the balloon in the medullary cavity of the femur; red filled squares, high-threshold (HT) neurons that responded to inflation of the balloon in the medullary cavity of the femur; red open squares, HT neurons that did not respond to inflation of the balloon in the medullary cavity of the femur; blue open triangle, low-threshold (LT) neurons that did not respond to inflation of the balloon in the medullary cavity of the femur. Bars indicate means of depth of recording sites of each type of the neurons from the dorsal surface of the spinal cord.

Figure 7.

Typical examples of recorded neurons. Typical examples of responses of wide-dynamic-range (WDR), high-threshold (HT), and low-threshold (LT) neurons to mechanical stimuli (a, b, and c). BR: brush stimulation; VF: stimulation using a 4-g von Frey filament; PI: pinch stimulation using an arterial clip with a force of 250 g/mm²; BA: sustained balloon inflation in the medullary cavity of the femur at 200 kPa.

Figure 8.

Cutaneous mechanical receptive fields of neurons that responded to intraosseous balloon inflation. Cutaneous mechanical receptive fields (RFs) of the dorsal horn neurons that responded to balloon inflation in the medullary cavity of the femur at 200 kPa. The edge of the low-threshold RF was defined as the area in which light touch stimulation with the tip of a 4-g von Frey filament elicited a response 50% of the time. The edge of the high-threshold RF was defined as the area in which high-intensity mechanical stimulation with a tungsten tip attached to a nylon filament (calibrated force of 25 g) elicited a response 50% of the time (see text). Wide-dynamic-range (WDR) neurons had low-threshold and high-thresholds RFs, while HT neurons had a high-threshold RF. Both WDR and HT neurons had cutaneous RFs at the lower back, lumbosacral region, and thigh. The schemas were made by overlaying the map of the RF area of each neuron repeatedly. HT: high-threshold.

Figure 9.

Relationship between intraosseous stimuli-evoked firing rate of neurons and intraosseous pressure of the femur. Responses of wide-dynamic-range (WDR) neurons to balloon inflation in the medullary cavity of the femur at pressures from 50 to 400 kPa. Data are expressed as means ± SEMs.

Figure 10.

Effects of morphine and naloxone on evoked responses of wide-dynamic-range neurons. Typical examples of evoked responses of a wide-dynamic-range (WDR) neuron to mechanical stimuli to the skin and balloon inflation before administration (control), 15 min after spinal administration of morphine, and 15 min after spinal administration of naloxone (a, b, and c). The cutaneous stimuli were applied in the most sensitive site of the receptive field. Mean changes in responses of WDR neurons (n = 5) to mechanical stimuli (d). BR: brush stimulation; PI: pinch stimulation using an arterial clip with a force of 250 g/mm²; BA: balloon inflation (200 kPa) in the medullary cavity of the femur. Data are expressed as means ± SEMs. *p < 0.01 versus control.