Role of Meningeal Mast Cells in Intrathecal Morphine–evoked Granuloma Formation

Abstract

BY histopathology and magnetic resonance imaging, intrathecal infusion of high concentrations of morphine in canine and ovine models had no effect on spinal parenchyma but led to an aseptic collection of inflammatory cells (granuloma).1–4Preclinical work shows that granulomas have several attributes, as follows: (1) the incidence is concentration dependent2–5and not observed after repeated bolus delivery6; (2) using serial magnetic resonance imaging in the dog, a mass may begin to form proximal to the catheter tip in an interval as short as 10 days, and termination of morphine resulted in a progressive reduction in size of the mass over the ensuing 14 days5; and (3) the mass arises from the dura–arachnoid layer of the meninges adjacent to the infusion catheter tip.3These observations are consistent with many human case series that have been reviewed.7–10An important question relates to the mechanism of this cellular accumulation. Bacterial presence has been reported but appears to be a rare, secondary, event (see Yaksh et al. 3and Lehmberg et al .11). Solutions are typically prepared to have cerebrospinal fluid–compatible osmolarity and pH.3,12,13Finally, infusion of vehicle (saline)2–4or a variety of drugs (see, e.g. , Sabbe et al .,14Yaksh et al. ,15and Chiari et al .16) fails to produce a granuloma in the dog. Substitution of saline for morphine infusate, after a mass has been established, reduces granuloma size.5Thus, the best characterized component appears to be morphine itself. We showed that several opioid molecules, including hydromorphone and methadone, resulted in intrathecal masses, whereas reduced, if any, granulomas were noted with high concentrations of fentanyl or the mu-opioid peptide [D-Ala,2N-MePhe,4Gly-ol5]-enkephalin, casting doubt as to whether this effect is opioid receptor mediated12(but see Johansen et al.).17Whether the morphine granuloma is prevented or reversed by opiate antagonism is unknown.Local accumulation of inflammatory cells arising from the meninges and its accessibility to the intrathecal infusate led us to consider that the mechanism might be a local drug effect on a meningeal target. The comparability of this phenomenon to plasma extravasation in other dural systems led us to consider the role of meningeal mast cells. Our hypothesis relating mast cell function to the morphine-induced granuloma is based on four considerations: (1) mast cells are distributed in dura–arachnoid18–20; (2) dural mast cell activation leads to local vasodilatation and release of chemoattractants that enhance cell migration21,22; (3) opiates degranulate cutaneous mast cells in skin but not reliably mast cells in lung, intestine, heart, or blood basophils23,24; and (4) because certain opiates (morphine) but not all (fentanyl) degranulate skin mast cells leading to dilation and plasma extravasation25–29and because the ability of an opiate antagonist to prevent such degranulation is controversial,30,31the role of opiate receptors in this phenomenon appears unlikely. This profile suggests similarities to that of intrathecal morphine–evoked granulomas and is consistent with the hypothesis that granulomas involve a mast cell contribution. This hypothesis led to a series of studies to characterize opiate effects in ex vivo and in vivo models on mast cell degranulation and the contribution of this action to the granuloma initiated by continuous intrathecal morphine in the canine model.All studies described in this article were accomplished under method protocols approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. There are two principal components to this series of studies: ex vivo and in vivo. These studies sought to determine the pharmacology of meningeal mast cell degranulation. For these studies, mixed-breed hounds (two male and three female animals, weighing approximately 25–35 kg) undergoing terminal experiments examining cardiac function were used.The animals were deeply anesthetized to the minimum of a surgical plane anesthesia sufficiently deep to perform a laminectomy (35 mg/kg sodium pentobarbital IV or 5–10 μg·kg−1·min−1propofol IV). The lumbar and thoracic vertebral bodies were exposed, cardiac arrest was initiated, and the dura was immediately harvested from the lumbar and thoracic levels. The dural fragments were immediately placed in an oxygenated iced Krebs–Ringer’s solution. The dural segments were cut into small fragments weighing approximately 10 mg. These were placed in individual Eppendorf tubes filled with Krebs–Ringer’s solution (1 ml) that were oxygenated by bubbling with 95% O2–5% CO2and warmed in a 37°C heating block. After an initial 20-min incubation, the solution was removed by aspiration and replaced. At this time, an aliquot of the test agent(s) was pipetted into each chamber. After a 30-min incubation, the solution was aspirated into individual containers and frozen until assay. The dural fragments were then gently blotted dry and weighed. The fragment was then placed in formalin and refrigerated. The dural fragment was mounted on a cryostat, and tangential 10-μm sections were taken and mounted on glass slides.Each incubation sample (100 μl) was assayed for histamine content. Histamine was analyzed using enzyme-linked immunosorbent assay (Research Diagnostics, Inc., Flanders, NJ). The limit of detection of this assay was approximately 0.3 ng/ml. The intraaasy and interassay precision was less than 11.0 and less than 12.0% coefficient of variation, respectively (3–30 ng/ml).Mast cells in the dural sections were stained with Alcian Blue or with naphthol AS-D chloroacetate esterase staining, a Fast Red staining technique, following methods provided by the manufacturer (Sigma-Aldrich, St. Louis, MO). Samples were surveyed under a microscope at 20× magnification. In each fragment, five stained sections were then examined at 60× magnification in a 100-μm field taken in the center of each fragment.To characterize the effects of intrathecal drug treatments on granuloma formation, groups of animals were prepared with chronic lumbar intrathecal catheters. They were then assigned to be entered into protocols. The groups and number of animals are listed in Table 1.Beagle dogs (Ridglan Farms Inc., Mt. Horeb, WI, or equivalent), 12 to 16 months of age and weighing approximately 9–16 kg, were housed individually in runs with wood shavings and given ad libitum access to food and water. Animals were adapted for a minimum of 10 days before surgery. A nylon vest for pump placement was placed on each dog 48 hours before intrathecal catheter placement for acclimation.Drugs were injected subcutaneously in a shaved region of the nape of the neck using a 25-gauge needle.Continuous intrathecal drug delivery was performed as follows:Male beagles weighing approximately 7–12 kg (n = 4) were anesthetized with IV propofol (5 μg·kg−1·min−1). Animals were intubated, and body temperature was maintained with an underbody heating pad and monitored continuously as described above. The chest and abdomen were shaven and surgically prepared. Intradermal injections of drug solutions were delivered in 50 μl at time 0 at 12–14 sites (six or seven per side). The injection sites were marked with ink. The diameter of the redness around each injection site was measured across its longest and narrowest axis and recorded without reference to drug treatment at time 0 and at 10, 30, and 60 min. Flare area immediately after injection and at intervals were calculated in square millimeters as an oval (3.14 × a × b , where a = half length of long axis and b = half length of short axis). Animals were used at 5-day intervals. This protocol was similar to that previously described to assess flare in dogs.35,36Each animal was used five to seven times.Preservative-free, U.S. Food and Drug Administration–approved formulations of morphine sulfate (Infumorph; Abbott, Abbott Park, IL), hydromorphone hydrochloride (Dilaudid-HP for Injection; Abbott), and saline vehicle (0.9% NaCl; Abbott) were used. Other opioids used were prepared from the powder. D/L-Methadone HCl and fentanyl HCl were obtained from the National Institute on Drug Abuse. The opiate antagonists naloxone HCl and naltrexone HCl, the mast cell degranulating Compound 48/80, and the mast cell stabilizers cromolyn sodium salt and nedocromil sodium were obtained from Sigma Chemical (St. Louis, MO). All drugs were prepared in saline (0.9%) unless stated otherwise. Doses and concentrations used are indicated in the text.Comparisons across treatment groups for granuloma size and for sensorimotor scores used the nonparametric Kruskal-Wallis test with post hoc comparisons to the saline treatment group using the Dunn multiple comparison test. For the cutaneous flare, comparisons were made across treatment using one-way ANOVA with post hoc comparison to the saline (vehicle control) group using the Dunnett multiple comparison test. For specific post hoc comparisons between the flare-inducing agent alone and with a co-treatment, multiple two-tailed t tests were performed, with a Bonferroni correction for alpha buildup performed for each set of tests. For analysis of thermal escape thresholds over time as a function of dose and treatment, a two-way repeated measures ANOVA was performed with post hoc comparisons using the Bonferroni test. To relate degree of sensorimotor deficits to granuloma size, we undertook a linear regression of sensorimotor score at sacrifice versus cross-sectional area of the granuloma. Group comparisons having critical values corresponding to P < 0.05 were considered to be statistically significant. The GraphPad Prism software package (v.4.0c for MAC OS X; GraphPad Software Inc., La Jolla, CA) was used for all analyses.Previous work has shown that intrathecal infusion of saline has no untoward histopathologic reactions through approximately 4 weeks (fig. 1). In contrast, morphine at 12 mg/day (e.g. , 12.5 mg/ml delivered at 960 μl/h) in the dog led to the development of prominent granulomas within 2 weeks in essentially all animals so infused.3,12Figure 1displays representative granulomas from three of the last 15 dogs receiving 12 mg/day morphine for approximately 28 days. Using ex vivo magnetic resonance imaging, volumetric reconstruction of the granuloma of four dogs that received 12 mg/day for greater than 24 days revealed calculated mass volumes of 0.42, 0.39, 0.31, and 0.36 cm3. As indicated by the typical volumetric reconstruction, the greatest girth of the mass was typically at the level of the catheter tip (fig. 2). In the animal imaged in figure 2, dual catheters had been placed to permit infusion and sampling. As indicated, the granuloma developed around the infusion catheter and was largest at the tip of the infusion catheter (e.g. , the drug delivery site). In the 15 dogs receiving intrathecal morphine (12 mg/day), the area of the granuloma at the level of largest girth was calculated. The median of the maximum area of the pericatheter mass was 9.5 mm2(interquartile range [IQR], 8.1–14.0 mm2). The corresponding median percentage of the cross-sectional area was 27.1% (IQR, 23.3–33.1%), emphasizing the compressive nature of these masses. The distribution of the cross-sectional area and percentage of the cross-sectional area of these masses is presented in figure 3and discussed further below. As reviewed previously,3examination of the granulomas revealed the presence of inflammatory cells with extensive immunoreactivity for CD68-IR macrophages and T cells (Leu4) (data not shown).Staining of cryostat sections of dural fragments with Alcian Blue or Fast Red revealed in the cervical, thoracic, and lumbar dura of a normal dog the significant presence of dense granule-containing profiles of mast cells (fig. 4A). Typically, in any given section, although some mast cells were found in the dura mater, the majority appeared to be present in the arachnoid layer. As shown in figure 4B, they frequently were found to distribute along the axis of the numerous small blood vessels present in the arachnoid layer. Harvest of dura from dogs that had received either no infusion (control) or approximately 28 days of saline vehicle revealed a large number of intact mast cell profiles. However, after 28 days of morphine (12 mg/day) and displaying a granuloma proximal to the catheter tip, proximal dura typically revealed relatively few intact profiles in the lumbar region. In contrast, examination of spinal dura harvested from upper thoracic (e.g. , T5–T10) and cervical (C2–C3) levels, where no granuloma was present, revealed significant numbers of intact granule-containing profiles (fig. 4C). Similar staining profiles were observed with naphthol AS-D chloroacetate esterase staining, a Fast Red staining technique (not shown).To determine the pharmacology of canine cutaneous mast cell degranulation, the flare-producing effects of intradermal injections of Compound 48/80 and several opiates were assessed in the anesthetized dog. Subcutaneous injection of saline produced at most a small bleb at the injection site that typically resolved within 10 min, with no incidence of reddening (flare). Figure 5Ashows the visual image of the concentration-dependent flare produced by side-by-side subcutaneous injections of 50 μl of increasing concentrations of Compound 48/80, morphine, or saline (vehicle). Figure 5Bsummarizes the flare areas produced by subcutaneous Compound 48/80 (0.1–10 mg/ml), morphine (0.01–10 mg/ml), and methadone (0.1–10 mg/ml). These agents produced a maximum increase in flare area by 30 min that typically disappeared over the next several hours. In contrast, no change was observed with fentanyl. Ordering of the magnitude revealed that at the concentrations used for morphine and methadone, effects were not from produced by the mast cell degranulation agent Compound 48/80 In contrast, fentanyl at the dose produced flare, although from was to of fentanyl were not as at these the multiple injections resulted in delivery of a dose to approximately and produced a However, no significant was noted in determine whether these effects were by mast cell degranulation, dogs were 60 min in with the mast cell stabilizers cromolyn mg/kg or nedocromil mg/kg As indicated in agents the flare produced by all In a a dose of cromolyn mg/kg the flare after Compound 48/80 and morphine (data not mg/kg the was examined on the flare produced by high concentrations of Compound 48/80 and morphine this dose prevented the observed flare produced by these agents (data not important question was whether these effects from the opiates were by a high dose of naloxone mg/kg given 30 min before the to the flare change produced by each of the agents (fig. The dose was at opiate receptor as the observed with the fentanyl was not the observations in vivo on the pharmacology of degranulation of mast cells as measured by the flare and to dural degranulation with cutaneous degranulation, we examined ex vivo histamine release from canine dural shown in figure dural fragments for 30 min with morphine resulted in a significant concentration-dependent degranulation of mast cells (fig. and an increase in histamine in the dural (fig. over the range of morphine concentrations of 10 to As indicated, morphine in this is at as as the release by of Compound 48/80, a mast the histamine release was by with cromolyn but not by Similar but studies examining meningeal histamine release were with other opioids at the As shown in 2, hydromorphone and methadone, but not to a concentration of significant in histamine release from saline and to observed with examination of the mast cell in the dural fragments after a 30-min incubation with vehicle revealed numerous mast cell profiles with dense staining (fig. of morphine resulted in a prominent change in of mast cell staining, with an increasing of the dural of cellular profiles with of degranulation (fig. following studies were performed to the in vivo antagonism by naltrexone of the effects of a dose of intrathecal morphine in the dog. The intrathecal infusion of morphine and 12 mg/day) resulted in an increase in thermal escape as with vehicle In a a injection of naltrexone prevented the increase over this interval (fig. determine the effects of opiate antagonism on intrathecal spinal granuloma formation, animals were injected with the dose of naltrexone mg/kg As indicated in figure in animals receiving 12 mg/day morphine for approximately 4 weeks with this dose of the (fig. and incidence of granuloma was not from that observed with morphine the median cross-sectional area in dogs receiving intrathecal morphine alone or with naltrexone was 9.5 8.1–14.0 and and were statistically from vehicle < determine the effects of a delivered mast cell on intrathecal spinal granuloma formation, animals received an intrathecal dose of 12 mg/day morphine with an of cromolyn infusion concentrations of 12 mg/day (n = These were based on the ex vivo work (fig. of cromolyn were observed to prevent mast cell degranulation. In this the incidence and the median area of the pericatheter masses in animals receiving intrathecal morphine (12 over an interval of days were by intrathecal cromolyn of five with median percentage of the cross-sectional area with 0 and were from the intrathecal group < but not from the saline group (fig. dogs received an intrathecal infusion of cromolyn (12 mg/day) In this no untoward effects on were and and staining showed no untoward (data not shown).To determine the effects of a delivered mast cell on intrathecal spinal granuloma formation, animals received an intrathecal delivery of 12 mg/day morphine and of mg/kg cromolyn (n = These were based on the in vivo degranulation work (fig. cromolyn was observed to prevent intradermal morphine–evoked mast cell degranulation. In this the median percentage of the cross-sectional area was animals receiving subcutaneous over an interval of days and were from the intrathecal group < but not from the saline group described of continuous infusion of 12 mg/day leads to an initial that is by an of the infusion concentration over days. the ensuing many animals receiving morphine but not vehicle that and an to to the and function were not sufficiently to Figure the sensorimotor score of all animals at the time of As indicated in figure the animals receiving morphine a median score of as with a median score of across all treatment groups revealed a significant but the morphine versus saline comparison was significant. We the score the percentage cross-sectional area of the granuloma and observed a regression although statistically 95% with was This suggests that the degree of observed with the of the granuloma with the compressive of the granuloma. several animals with significant showed no deficits at the time of This was a that we intrathecal morphine a accumulation of inflammatory cells from the meningeal We that dural mast cell degranulation is a between intrathecal opiates and the the dog, intrathecal infusion of approximately concentrations of morphine, or methadone, but not in an intrathecal drug effect along with of naltrexone in the present work to the a opioid of naltrexone might be to a we the naltrexone and its have of several with that of the see and has opiate antagonist that may for the long of by the the dose used resulted in a significant antagonism of the effects produced by the dose for an of in an of 16 of receptor In we showed that repeated bolus morphine resulted in no that between morphine injection were to the agent and prevent granuloma formation, a by antagonist we that size be a of granuloma and this was in greater in the naltrexone we that the effects of intrathecal morphine are not on a opiate cells are distributed in spinal with local arachnoid meningeal mast cells are to degranulation by the local activation and including and chemoattractants for T and of and the meningeal and is present in large in all mast contribution to plasma extravasation and cell in dura led us to consider the role of meningeal mast cells in the intrathecal accumulation of inflammatory cells the degranulate mast cells and release the skin where mast cells are in a local cutaneous , a on the pharmacology of this cutaneous effect shows that although a variety of opioids produce this effect is not with an opiate subcutaneous injection of some opiate but not other agents in histamine in a that is to opiate antagonism but prevented by (e.g. , cromolyn and this series and of opiate antagonism to the effects of dural or cutaneous mast cell degranulation suggests that opiate receptors at most a as shown meningeal mast cells are by morphine, but not by and this effect is not by concentrations of naloxone that are than to effects of opiate receptor activation in in between dural and cutaneous mast cell activation is in in contrast, mast cells from the heart, lung, and are reported to not a to between dura and cutaneous mast cells may the that dura and skin from the and and mast cells similar opiates to degranulate mast We several delivery of cromolyn or subcutaneous cromolyn resulted in a reduction in the incidence and cross-sectional area of granulomas in dogs receiving continuous infusion of a dose of intrathecal cromolyn in prevented dural mast cell degranulation noted after prevent degranulation by a variety of from has other including (1) of in mast and P and (3) plasma role these other in the observed is not studies the of the cromolyn effect and the of mast cell stabilizers to the role of the be that mast cells may be an mechanism several although granuloma is considered to be two observations are (1) is an increasing incidence of and in these masses, and (2) using intrathecal a to a granuloma (see et al. for this is not an effect to be Our work to the of this The present be considered as a contribution to a of the role of meningeal mast cells. The not other that may not mast cells. the canine work a mast cell effect with We not whether mast cell degranulation a granuloma. an effect of cromolyn be as a of the drug by either or the used. such and is to the of this or any to of University of the to the of the meningeal to the that in the effects of opiates in degranulating mast and the role of mast cells. on The this article to The of of and for to these