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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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Histology verifies MR imaging findings with perineural iron-laden macrophages demonstrated only in animals injected with USPIOs without minocycline. Paraffin sections were prepared from the distal segment of the nerve, just proximal to the ligature, and stained with (A, C, E, G) hematoxylin and eosin (H&E) to provide morphological detail and with (B, D, F, H) Perl’s stain, which stains iron blue. (A, B) Control (−USPIO) longitudinal sections through the neuroma and surrounding perineural tissue harvested from the site of the neuroma show no iron staining. (C-F) Tissues harvested from SNI animals injected with USPIOs. At 200X magnification, the axial section and the longitudinal section (C, D) and an axial section (E, F) from a different animal show areas of increased iron staining in the perineural tissues. These iron containing cells demonstrate histologic characteristics of macrophages. At 200x magnification, iron and H&E stained axial sections from animals treated with minocycline and USPIOs (+MINO-USPIO) show localization of iron containing cells to the perineural tissue, albeit to a qualitatively lesser extent than the –MINO+USPIO treated sections.
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Figure 4: Histology verifies MR imaging findings with perineural iron-laden macrophages demonstrated only in animals injected with USPIOs without minocycline. Paraffin sections were prepared from the distal segment of the nerve, just proximal to the ligature, and stained with (A, C, E, G) hematoxylin and eosin (H&E) to provide morphological detail and with (B, D, F, H) Perl’s stain, which stains iron blue. (A, B) Control (−USPIO) longitudinal sections through the neuroma and surrounding perineural tissue harvested from the site of the neuroma show no iron staining. (C-F) Tissues harvested from SNI animals injected with USPIOs. At 200X magnification, the axial section and the longitudinal section (C, D) and an axial section (E, F) from a different animal show areas of increased iron staining in the perineural tissues. These iron containing cells demonstrate histologic characteristics of macrophages. At 200x magnification, iron and H&E stained axial sections from animals treated with minocycline and USPIOs (+MINO-USPIO) show localization of iron containing cells to the perineural tissue, albeit to a qualitatively lesser extent than the –MINO+USPIO treated sections.

Mentions: Histology of these harvested sciatic nerve specimens confirmed the presence of iron-laden macrophages at the nerve injury site only in the group of injured rats that received USPIOs without minocycline treatment (Figure 4C-F). By comparison, rats that received minocycline had qualitatively fewer iron-laden macrophages at the site of nerve injury as determined by Perl’s staining (Figure 4G, H).


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)

Histology verifies MR imaging findings with perineural iron-laden macrophages demonstrated only in animals injected with USPIOs without minocycline. Paraffin sections were prepared from the distal segment of the nerve, just proximal to the ligature, and stained with (A, C, E, G) hematoxylin and eosin (H&E) to provide morphological detail and with (B, D, F, H) Perl’s stain, which stains iron blue. (A, B) Control (−USPIO) longitudinal sections through the neuroma and surrounding perineural tissue harvested from the site of the neuroma show no iron staining. (C-F) Tissues harvested from SNI animals injected with USPIOs. At 200X magnification, the axial section and the longitudinal section (C, D) and an axial section (E, F) from a different animal show areas of increased iron staining in the perineural tissues. These iron containing cells demonstrate histologic characteristics of macrophages. At 200x magnification, iron and H&E stained axial sections from animals treated with minocycline and USPIOs (+MINO-USPIO) show localization of iron containing cells to the perineural tissue, albeit to a qualitatively lesser extent than the –MINO+USPIO treated sections.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472277&req=5

Figure 4: Histology verifies MR imaging findings with perineural iron-laden macrophages demonstrated only in animals injected with USPIOs without minocycline. Paraffin sections were prepared from the distal segment of the nerve, just proximal to the ligature, and stained with (A, C, E, G) hematoxylin and eosin (H&E) to provide morphological detail and with (B, D, F, H) Perl’s stain, which stains iron blue. (A, B) Control (−USPIO) longitudinal sections through the neuroma and surrounding perineural tissue harvested from the site of the neuroma show no iron staining. (C-F) Tissues harvested from SNI animals injected with USPIOs. At 200X magnification, the axial section and the longitudinal section (C, D) and an axial section (E, F) from a different animal show areas of increased iron staining in the perineural tissues. These iron containing cells demonstrate histologic characteristics of macrophages. At 200x magnification, iron and H&E stained axial sections from animals treated with minocycline and USPIOs (+MINO-USPIO) show localization of iron containing cells to the perineural tissue, albeit to a qualitatively lesser extent than the –MINO+USPIO treated sections.
Mentions: Histology of these harvested sciatic nerve specimens confirmed the presence of iron-laden macrophages at the nerve injury site only in the group of injured rats that received USPIOs without minocycline treatment (Figure 4C-F). By comparison, rats that received minocycline had qualitatively fewer iron-laden macrophages at the site of nerve injury as determined by Perl’s staining (Figure 4G, H).

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