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In vivo USPIO magnetic resonance imaging shows that minocycline mitigates macrophage recruitment to a peripheral nerve injury.

Ghanouni P, Behera D, Xie J, Chen X, Moseley M, Biswal S - Mol Pain (2012)

Bottom Line: Minocycline has proven anti-nociceptive effects, but the mechanism by which minocycline delays the development of allodynia and hyperalgesia after peripheral nerve injury remains unclear.Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p < 0.011).Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/-0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/-4.5%) and minocycline-treated group that did not receive USPIOs (41.2+/-2.3%) (p < 0.04).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiology, Molecular Imaging Program at Stanford (MIPS) Stanford, Stanford University School of Medicine, California, USA.

ABSTRACT

Background: Minocycline has proven anti-nociceptive effects, but the mechanism by which minocycline delays the development of allodynia and hyperalgesia after peripheral nerve injury remains unclear. Inflammatory cells, in particular macrophages, are critical components of the response to nerve injury. Using ultrasmall superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI) to monitor macrophage trafficking, the purpose of this project is to determine whether minocycline modulates macrophage trafficking to the site of nerve injury in vivo and, in turn, results in altered pain thresholds.

Results: Animal experiments were approved by Stanford IACUC. A model of neuropathic pain was created using the Spared Nerve Injury (SNI) model that involves ligation of the left sciatic nerve in the left thigh of adult Sprague-Dawley rats. Animals with SNI and uninjured animals were then injected with/without USPIOs (300 μmol/kg i.v.) and with/without minocycline (50 mg/kg i.p.). Bilateral sciatic nerves were scanned with a volume coil in a 7 T magnet 7 days after USPIO administration. Fluid-sensitive MR images were obtained, and ROIs were placed on bilateral sciatic nerves to quantify signal intensity. Pain behavior modulation by minocycline was measured using the Von Frey filament test. Sciatic nerves were ultimately harvested at day 7, fixed in 10% buffered formalin and stained for the presence of iron oxide-laden macrophages. Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p < 0.011). Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/-0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/-4.5%) and minocycline-treated group that did not receive USPIOs (41.2+/-2.3%) (p < 0.04). Histology of harvested sciatic nerve specimens confirmed the presence USPIOs at the nerve injury site in the SNI group without minocycline treatment.

Conclusion: Animals with neuropathic pain in the left hindpaw show increased trafficking of USPIO-laden macrophages to the site of sciatic nerve injury. Minocycline to retards the migration of macrophages to the nerve injury site, which may partly explain its anti-nociceptive effects. USPIO-MRI is an effective in vivo imaging tool to study the role of macrophages in the development of neuropathic pain.

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Percent increase in relative nerve signal intensity in the setting of minocycline and USPIO treatment. The signal intensity of the injured left and uninjured right sciatic nerve proximal to the site of injury was normalized to that of the adjacent muscle. Asterisks denote injured left nerve treatment groups that were significantly different from the untreated control animals (Bonferroni’s post hoc multiple comparison test, p < 0.05 for +USPIO versus +MINO +USPIO; p < 0.001 for +MINO +USPIO and for +USPIO versus -MINO -USPIO; no difference between +MINO and –MINO -USPIO). The signal intensity of uninjured right nerve was only mildly higher than that of surrounding muscle (3 to 13%), which was independent of minocycline and/or USPIO administration. Data represent the averages and standard deviations of three animals in each group.
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Figure 3: Percent increase in relative nerve signal intensity in the setting of minocycline and USPIO treatment. The signal intensity of the injured left and uninjured right sciatic nerve proximal to the site of injury was normalized to that of the adjacent muscle. Asterisks denote injured left nerve treatment groups that were significantly different from the untreated control animals (Bonferroni’s post hoc multiple comparison test, p < 0.05 for +USPIO versus +MINO +USPIO; p < 0.001 for +MINO +USPIO and for +USPIO versus -MINO -USPIO; no difference between +MINO and –MINO -USPIO). The signal intensity of uninjured right nerve was only mildly higher than that of surrounding muscle (3 to 13%), which was independent of minocycline and/or USPIO administration. Data represent the averages and standard deviations of three animals in each group.

Mentions: We then sought to monitor minocycline’s effect on macrophages by labeling these cells in vivo using ultrasmall superparamagnetic iron oxide particles. After intravenous injection, these particles are phagocytosed by reticuloendothelial cells, including macrophages [2]. Delivery of the particles was confirmed by abdominal MR imaging that demonstrated a dramatic decline in the T2 weighted signal intensity of the liver and spleen in animals injected with USPIOs. On FIESTA MR, the injured sciatic nerve was enlarged compared to the uninjured nerve, and demonstrated increased signal intensity relative to muscle (relative MR signal intensity: 37 ± 1%, Figure 2A). Minocycline treatment alone did not alter the increased signal intensity of the injured nerve (43 ± 3%). However, with USPIO administration, the injured nerves became nearly isointense to the surrounding muscle (7 ± 3%, Figure 2B), suggesting that USPIO-laden cells had migrated to the site of injury. While this decline in signal was specific to animals that received USPIO injections, we further assessed for post-operative hematomas as a possible confounding source of iron-laden cells. No hematoma was evident at the site of injury during the course of imaging. In addition, copious saline washes of the surgical site did not alter the MR signal properties of the injured nerve (data not shown). Administration of minocycline significantly reduced the USPIO effect (nerve signal relative to muscle: 17 ± 5%, Figure 2C, Figure 3, p < 0.05). In order to determine the etiology of the decreased MR signal of the injured nerve in rats treated with USPIOs, rat sciatic nerves were harvested and examined. Of note, the signal intensity of uninjured right nerve was only mildly higher than that of surrounding muscle (3 to 7%), which was independent of minocycline and/or USPIO administration (Figures 2F, Figure 3).


In vivo USPIO magnetic resonance imaging shows that minocycline mitigates macrophage recruitment to a peripheral nerve injury.

Ghanouni P, Behera D, Xie J, Chen X, Moseley M, Biswal S - Mol Pain (2012)

Percent increase in relative nerve signal intensity in the setting of minocycline and USPIO treatment. The signal intensity of the injured left and uninjured right sciatic nerve proximal to the site of injury was normalized to that of the adjacent muscle. Asterisks denote injured left nerve treatment groups that were significantly different from the untreated control animals (Bonferroni’s post hoc multiple comparison test, p < 0.05 for +USPIO versus +MINO +USPIO; p < 0.001 for +MINO +USPIO and for +USPIO versus -MINO -USPIO; no difference between +MINO and –MINO -USPIO). The signal intensity of uninjured right nerve was only mildly higher than that of surrounding muscle (3 to 13%), which was independent of minocycline and/or USPIO administration. Data represent the averages and standard deviations of three animals in each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Percent increase in relative nerve signal intensity in the setting of minocycline and USPIO treatment. The signal intensity of the injured left and uninjured right sciatic nerve proximal to the site of injury was normalized to that of the adjacent muscle. Asterisks denote injured left nerve treatment groups that were significantly different from the untreated control animals (Bonferroni’s post hoc multiple comparison test, p < 0.05 for +USPIO versus +MINO +USPIO; p < 0.001 for +MINO +USPIO and for +USPIO versus -MINO -USPIO; no difference between +MINO and –MINO -USPIO). The signal intensity of uninjured right nerve was only mildly higher than that of surrounding muscle (3 to 13%), which was independent of minocycline and/or USPIO administration. Data represent the averages and standard deviations of three animals in each group.
Mentions: We then sought to monitor minocycline’s effect on macrophages by labeling these cells in vivo using ultrasmall superparamagnetic iron oxide particles. After intravenous injection, these particles are phagocytosed by reticuloendothelial cells, including macrophages [2]. Delivery of the particles was confirmed by abdominal MR imaging that demonstrated a dramatic decline in the T2 weighted signal intensity of the liver and spleen in animals injected with USPIOs. On FIESTA MR, the injured sciatic nerve was enlarged compared to the uninjured nerve, and demonstrated increased signal intensity relative to muscle (relative MR signal intensity: 37 ± 1%, Figure 2A). Minocycline treatment alone did not alter the increased signal intensity of the injured nerve (43 ± 3%). However, with USPIO administration, the injured nerves became nearly isointense to the surrounding muscle (7 ± 3%, Figure 2B), suggesting that USPIO-laden cells had migrated to the site of injury. While this decline in signal was specific to animals that received USPIO injections, we further assessed for post-operative hematomas as a possible confounding source of iron-laden cells. No hematoma was evident at the site of injury during the course of imaging. In addition, copious saline washes of the surgical site did not alter the MR signal properties of the injured nerve (data not shown). Administration of minocycline significantly reduced the USPIO effect (nerve signal relative to muscle: 17 ± 5%, Figure 2C, Figure 3, p < 0.05). In order to determine the etiology of the decreased MR signal of the injured nerve in rats treated with USPIOs, rat sciatic nerves were harvested and examined. Of note, the signal intensity of uninjured right nerve was only mildly higher than that of surrounding muscle (3 to 7%), which was independent of minocycline and/or USPIO administration (Figures 2F, Figure 3).

Bottom Line: Minocycline has proven anti-nociceptive effects, but the mechanism by which minocycline delays the development of allodynia and hyperalgesia after peripheral nerve injury remains unclear.Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p < 0.011).Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/-0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/-4.5%) and minocycline-treated group that did not receive USPIOs (41.2+/-2.3%) (p < 0.04).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Radiology, Molecular Imaging Program at Stanford (MIPS) Stanford, Stanford University School of Medicine, California, USA.

ABSTRACT

Background: Minocycline has proven anti-nociceptive effects, but the mechanism by which minocycline delays the development of allodynia and hyperalgesia after peripheral nerve injury remains unclear. Inflammatory cells, in particular macrophages, are critical components of the response to nerve injury. Using ultrasmall superparamagnetic iron oxide-magnetic resonance imaging (USPIO-MRI) to monitor macrophage trafficking, the purpose of this project is to determine whether minocycline modulates macrophage trafficking to the site of nerve injury in vivo and, in turn, results in altered pain thresholds.

Results: Animal experiments were approved by Stanford IACUC. A model of neuropathic pain was created using the Spared Nerve Injury (SNI) model that involves ligation of the left sciatic nerve in the left thigh of adult Sprague-Dawley rats. Animals with SNI and uninjured animals were then injected with/without USPIOs (300 μmol/kg i.v.) and with/without minocycline (50 mg/kg i.p.). Bilateral sciatic nerves were scanned with a volume coil in a 7 T magnet 7 days after USPIO administration. Fluid-sensitive MR images were obtained, and ROIs were placed on bilateral sciatic nerves to quantify signal intensity. Pain behavior modulation by minocycline was measured using the Von Frey filament test. Sciatic nerves were ultimately harvested at day 7, fixed in 10% buffered formalin and stained for the presence of iron oxide-laden macrophages. Behavioral measurements confirmed the presence of allodynia in the neuropathic pain model while the uninjured and minocycline-treated injured group had significantly higher paw withdrawal thresholds (p < 0.011). Decreased MR signal is observed in the SNI group that received USPIOs (3.3+/-0.5%) compared to the minocycline-treated SNI group that received USPIOs (15.2+/-4.5%) and minocycline-treated group that did not receive USPIOs (41.2+/-2.3%) (p < 0.04). Histology of harvested sciatic nerve specimens confirmed the presence USPIOs at the nerve injury site in the SNI group without minocycline treatment.

Conclusion: Animals with neuropathic pain in the left hindpaw show increased trafficking of USPIO-laden macrophages to the site of sciatic nerve injury. Minocycline to retards the migration of macrophages to the nerve injury site, which may partly explain its anti-nociceptive effects. USPIO-MRI is an effective in vivo imaging tool to study the role of macrophages in the development of neuropathic pain.

Show MeSH
Related in: MedlinePlus