- Email: [email protected]
The quantification of cutaneous autonomic pilomotor innervation
106
Abstracts / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133
ethonium (Hex; 25 mg/kg). Drugs were delivered to the left iliac artery via a retrograde catheter insertion in the right iliac artery to the aorta bifurcation. Following Hex, mean arterial pressure (baseline: 83 ± 18, HEX: 57 ± 3 mm Hg) and blood flow velocity (baseline: 28 ± 5, HEX: 20 ± 4 mm/s) (n = 6) were reduced below baseline levels for about 30 min after which the dilatory response in the sciatic nerve appeared. Specifically, the index of conductance (velocity/MAP expressed at % baseline) increased from 103 ± 35 mm/s/mm Hg after 30 min of Hex to 127 ± 39 mm/s/mm Hg in the following 60 min of Hex (p < 0.05). In a second group of animals, using the 30-min of Hex as the new baseline, with a top-up of Hex, a 10-min infusion of NPY (Y1 agonist, 15 μg/kg) minimized the late dilatory response (n = 6, Hex baseline: 99 ± 15, NPY: 104 ± 11 mm/s/mm Hg; NS). This NPYinduced attenuation of the late dilation was, in turn, minimized by BIBP (Y1 antagonist, 100 μg/kg) (n = 4, Hex baseline: 73 ± 12, BIBP: 89 ± 14 mm/s/mm Hg). Neither NPY (n = 7) nor BIBP3226 (n = 4) infusions without Hex had any effect on sciatic nerve arterial conductance. Therefore, the late dilation following Hex was reversed by Y1 receptor activation suggesting some level of sympathetic control over sciatic nerve blood flow. While this control was evident with ganglionic blockade, it was not apparent under baseline conditions raising the possibility that Y1R neurovascular regulation is not obligatory. Keywords: sciatic nerve, nerve blood flow, doppler ultrasound, Y1 receptor, wistar Kyoto rats Financial support: Supported by Canadian Institutes of Health Research.
doi:10.1016/j.autneu.2011.05.184
P.170 The Laser Doppler Imaging axon-reflex flare area/latency ratio: A novel measure of neurovascular C-fiber function T. Siepmann, B.M.W. Illigens, R. Freeman, C.H. Gibbons (Beth Israel Deaconess Medical Center, Harvard Medical School - Neurology, United States) Background/aims: Neurovascular function is assessed by Laser Doppler Flowmetry (LDF) or Laser Doppler Imaging (LDI) of axonreflex mediated blood flow. There is limited clinical utility for LDF because of high variability while LDI has no optimally defined image analysis technique for data interpretation. This study reports a novel LDI thresholding and analysis technique to quantify neurovascular dysfunction using a capsaicin model of neuropathy. Methods: Eighteen healthy subjects ages 21 to 27 underwent occlusive application of 0.1% capsaicin cream on the lateral thigh over 48 h with placebo randomly applied to the opposite thigh. Axonreflex blood flow was induced through acetylcholine iontophoresis. A novel regression analysis technique was used to determine the optimal LDI image thresholding level. LDI was performed at baseline (day −2) and on days 0, 7, 14, 21, 28 post-application. The axonreflex flare response was analyzed for flare area spread and latency to maximal spread. Results: The optimal thresholding level was 425 PU. The axon-reflex flare area was reduced in capsaicin treated skin compared to placebo (p < 0.01) on all days. The maximal difference in flare area was immediately post-capsaicin application (decrease from 974 ± 378 mm2 to 495 ± 307 mm2). The flare latency increased from 354±
59 to 735 ± 465 s immediately after capsaicin application (p< 0.01) but was unchanged on the following evaluation days. The area/latency ratio decreased after capsaicin application (p< 0.01) on all evaluation days with maximal attenuation immediately post-application (decrease from 2.7 ± 1.0 to 1.0 ± 0.9 mm2/s) and correlated with nerve fiber density. Conclusions: Neurovascular dysfunction in capsaicin induced neuropathy is detected by decreased LDI axon-reflex flare area and by reduced latency/area ratio using an optimal perfusion threshold of 425 PU. The flare latency-area ratio provides a composite measure of axonal C-fiber function that correlates with epidermal nerve fiber density and improves the utility of LDI in assessing neurovascular function. Keywords: Laser Doppler Imaging, vasomotor axon-reflex, capsaicin, Flare response, small fiber neuropathy Financial support: NIH, Langer Family Foundation.
doi:10.1016/j.autneu.2011.05.185
P.171 The quantification of cutaneous autonomic pilomotor innervation C.H. Gibbons, N. Wang, A. Hovaguimian, B.M.W. Illigens, R. Freeman (Beth Israel Deaconess Medical Center, Harvard Medical School Neurology, United States) Background: Our objective in this study is to define the topography of pilomotor nerve fiber density PNFD) in clinically relevant sites, to demonstrate the effectiveness of both dopamine β-hydroxylase (DβH) and tyrosine hydroxylase (TH) to stain for sympathetic adrenergic fibers, and determine the pilomotor nerve fiber density (PNFD) in healthy control subjects and individuals with diabetes using standard light microscopy PGP stained tissue sections. Methods: Thirty-four control and 46 subjects with clinical evidence of diabetic neuropathy were examined and punch skin biopsies at the distal leg, distal thigh, proximal thigh and forearm were obtained. Nerve fibers were labeled with PGP 9.5, TH, DβH, TH and VIP. Images were captured on a confocal microscope in 3 μm optical sections, or labeled by PGP 9.5 for light microscopy sections. Three blinded observers quantified nerve fiber density by counting fibers crossing perpendicular to the axis of the pilomotor muscle. Distribution of nerve fibers by location, subtype and inter-rater reliability was determined. Results: Subjects with neuropathy had reduced pilomotor nerve fiber density (PNFD) compared to controls at the distal leg (69 ± 9 vs 82 ± 16 fibers/mm; p < 0.05), distal thigh (64 ± 14 vs 106 ± 25 fibers/mm; p < 0.01), proximal thigh (85 ± 18 vs. 110 ± 19 fibers/mm; p < 0.05) and forearm (68 ± 12 vs 103 ± 24 fibers/mm; p < 0.01). There was 100% overlap between TH and DβH fibers in arrector pili muscles. The intraclass correlation coefficient was 0.89 (p < 0.001) for interobserver reliability. Conclusion: Quantification of PNFD, using both confocal and light microscopy image analysis techniques, differentiates a group of patients with neuropathy from healthy control subjects. Our results confirm a recently reported method to quantitate PNFD (Nolano
Abstracts / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133
Neurology 2010), expand the technique to light microscopy, use TH and DβH to stain for adrenergic fibers and identify the proximal to distal gradient in diabetic neuropathy. The high interclass correlation coefficients between reviewers confirm that our results are reproducible.
107
Keywords: spinal cord injury, hypertension, plasticity, pain, blood pressure Financial support: Heart and Stroke Foundation of BC and the Yukon (of Canada).
Keywords: Pilomotor, neuropathy, sympathetic, adrenergic, diabetes doi:10.1016/j.autneu.2011.05.187 Financial support: NIH K23NS050209. P.173 Transient changes in BDNF parallel plasticity observed in the IML following distal axon injury doi:10.1016/j.autneu.2011.05.186 A.P. Coulibaly, B.F. Walsh, L.G. Isaacson(Miami University - Zoology, United States) P.172 The role of capsaicin-sensitive sensory neurons in autonomic dysreflexia in the spinal cord injured-rat L.M. Ramer (University of British Columbia -International Collaboration on Repair Discoveries, Canada), A.P. van Stolk (UBC -ICORD, Canada), J.A. Inskip (Simon Fraser University -Biomedical Physiology and Kinesiology, Canada), M.S. Ramer (University of British Columbia International Collaboration on Repair Discoveries, Canada), A.V. Krassioukov (University of British Columbia -Physical Medicine and Rehabilitation, Canada) Background: A subpopulation of sensory neurons express the transient receptor potential cation channel subfamily V member 1 (TRPV1). It is activated by heat (>43 °C), low pH, and capsaicin (the pungent ingredient in hot chili peppers), and has emerged as a promising target for the treatment of pain. Both pain and autonomic dysreflexia that develop in the wake of SCI have been attributed to maladaptive sensory plasticity. We hypothesize that TRPV-1-positive afferents contribute to the development of autonomic dysreflexia (AD). Methods: Wistar rats (300 g) received a complete transection of the spinal cord at the third (T3) or tenth (T10) thoracic segment or sham injury (durotomy without SCI). After 1–12 weeks, dorsal root ganglia (DRGs) and spinal cords were harvested and analyzed immunohistochemically to examine soma size of TRPV1-positive afferents and density of their central projections. A subset of rats received vehicle or capsaicin via intrathecal injection at 28 days; at 30 days, the severity of AD was assessed in these animals, by recording intra-arterial blood pressure during colo-rectal distension (CRD). Results: TRPV-1-positive nociceptors exhibited hypertrophy after T3 SCI. Nociceptor hypertrophy only occurred in DRGs below the level of SCI, and was more pronounced after high thoracic (T3) SCI. Hypertrophy was pronounced in DRGs far distal to SCI, and occurred in DRGs that contained both somatic (L4,L5) and visceral (L6,S1) afferents. TRPV-1-expressing nociceptors also appeared to sprout within the lumbar dorsal horn: the area occupied by TRPV-1-positive axons was increased one month after SCI. CRD-evoked hypertension was less pronounced in rats treated with capsaicin than in vehicletreated animals. Conclusion: TRPV-1-expressing nociceptors respond to SCI by undergoing somal hypertrophy and expanding their central terminals in the lumbar dorsal horn. Selective elimination of the central projections of these nociceptors reduces the severity of AD in rats with SCI. We are now examining the intriguing possibility that spontaneous activity in these neurons may contribute to the development of AD after SCI.
The neurotrophic regulation of sympathetic preganglionic neurons located in the intermediolateral cell column (IML) is poorly understood. We previously reported transient changes in the adult IML following injury to the cervical sympathetic trunk (CST), the distal axons entering the superior cervical ganglion (SCG). At 1 week following CST transection, activated astrocytes and microglia and increased numbers of trkB immunoreactive (− ir) oligodendrocytes populated the IML at levels C8-T1. This glial plasticity was paralleled by a decrease in choline acetyltransferase (ChAT)-ir neurons. Though ChAT was lost, neurons were present and expressed the injury marker activating transcription factor (ATF)-3. At 3 weeks, glial activation was absent, ChAT-ir neurons showed recovery, and ATF3-ir neurons declined to 13% of IML neurons. The number and appearance of ChAT-ir neurons were similar to controls at 10 weeks following injury. The changes in trkB suggested a role for the neurotrophin brain derived neurotrophic factor (BDNF) during this period of plasticity and provided the basis for the present study where we used western blot analysis to investigate BDNF levels in the spinal cord at 1 week, 3 weeks, and 10 weeks following CST transection. At 1 week following injury, when glial cells respond and ChAT is decreased, the 24 kDa isoform showed a significant decrease in all cases and the 37 kDa isoform was decreased in 4 of the 5 cases examined. At 21 days, when gliosis has subsided and ChAT expression is recovering, the 37 kDa form was significantly increased. BDNF returned to control values at 10 weeks following injury. The transient changes in BDNF may play a role in the IML neuroplasticity observed following injury. The BDNF protein in the cord is likely produced locally since the transected axons do not reinnervate the SCG by 10 weeks and could not derive BDNF from their SCG target. Keywords: sympathetic preganglionic neuron, neurotrophin regulation, choline acetyltransferase, trkB Financial support: NIHNS051206 to LGI, Undergraduate Research grant to BFW.
doi:10.1016/j.autneu.2011.05.188
P.174 Spectral analysis of cardiovascular control after spinal cord injury in rats: Effect of time post-injury J.A. Inskip (Simon Fraser University/University of British Columbia Biomedical Physiology and Kinesiology/International Collaboration on Repair Discoveries, Canada), L.M. Ramer (University of British Columbia -International Collaboration on Repair Discoveries/Zoology, Canada), M.S. Ramer (University of British Columbia -International Collaboration on Repair Discoveries/Zoology, Canada), A.V. Krassiou-
Abstracts / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133
ethonium (Hex; 25 mg/kg). Drugs were delivered to the left iliac artery via a retrograde catheter insertion in the right iliac artery to the aorta bifurcation. Following Hex, mean arterial pressure (baseline: 83 ± 18, HEX: 57 ± 3 mm Hg) and blood flow velocity (baseline: 28 ± 5, HEX: 20 ± 4 mm/s) (n = 6) were reduced below baseline levels for about 30 min after which the dilatory response in the sciatic nerve appeared. Specifically, the index of conductance (velocity/MAP expressed at % baseline) increased from 103 ± 35 mm/s/mm Hg after 30 min of Hex to 127 ± 39 mm/s/mm Hg in the following 60 min of Hex (p < 0.05). In a second group of animals, using the 30-min of Hex as the new baseline, with a top-up of Hex, a 10-min infusion of NPY (Y1 agonist, 15 μg/kg) minimized the late dilatory response (n = 6, Hex baseline: 99 ± 15, NPY: 104 ± 11 mm/s/mm Hg; NS). This NPYinduced attenuation of the late dilation was, in turn, minimized by BIBP (Y1 antagonist, 100 μg/kg) (n = 4, Hex baseline: 73 ± 12, BIBP: 89 ± 14 mm/s/mm Hg). Neither NPY (n = 7) nor BIBP3226 (n = 4) infusions without Hex had any effect on sciatic nerve arterial conductance. Therefore, the late dilation following Hex was reversed by Y1 receptor activation suggesting some level of sympathetic control over sciatic nerve blood flow. While this control was evident with ganglionic blockade, it was not apparent under baseline conditions raising the possibility that Y1R neurovascular regulation is not obligatory. Keywords: sciatic nerve, nerve blood flow, doppler ultrasound, Y1 receptor, wistar Kyoto rats Financial support: Supported by Canadian Institutes of Health Research.
doi:10.1016/j.autneu.2011.05.184
P.170 The Laser Doppler Imaging axon-reflex flare area/latency ratio: A novel measure of neurovascular C-fiber function T. Siepmann, B.M.W. Illigens, R. Freeman, C.H. Gibbons (Beth Israel Deaconess Medical Center, Harvard Medical School - Neurology, United States) Background/aims: Neurovascular function is assessed by Laser Doppler Flowmetry (LDF) or Laser Doppler Imaging (LDI) of axonreflex mediated blood flow. There is limited clinical utility for LDF because of high variability while LDI has no optimally defined image analysis technique for data interpretation. This study reports a novel LDI thresholding and analysis technique to quantify neurovascular dysfunction using a capsaicin model of neuropathy. Methods: Eighteen healthy subjects ages 21 to 27 underwent occlusive application of 0.1% capsaicin cream on the lateral thigh over 48 h with placebo randomly applied to the opposite thigh. Axonreflex blood flow was induced through acetylcholine iontophoresis. A novel regression analysis technique was used to determine the optimal LDI image thresholding level. LDI was performed at baseline (day −2) and on days 0, 7, 14, 21, 28 post-application. The axonreflex flare response was analyzed for flare area spread and latency to maximal spread. Results: The optimal thresholding level was 425 PU. The axon-reflex flare area was reduced in capsaicin treated skin compared to placebo (p < 0.01) on all days. The maximal difference in flare area was immediately post-capsaicin application (decrease from 974 ± 378 mm2 to 495 ± 307 mm2). The flare latency increased from 354±
59 to 735 ± 465 s immediately after capsaicin application (p< 0.01) but was unchanged on the following evaluation days. The area/latency ratio decreased after capsaicin application (p< 0.01) on all evaluation days with maximal attenuation immediately post-application (decrease from 2.7 ± 1.0 to 1.0 ± 0.9 mm2/s) and correlated with nerve fiber density. Conclusions: Neurovascular dysfunction in capsaicin induced neuropathy is detected by decreased LDI axon-reflex flare area and by reduced latency/area ratio using an optimal perfusion threshold of 425 PU. The flare latency-area ratio provides a composite measure of axonal C-fiber function that correlates with epidermal nerve fiber density and improves the utility of LDI in assessing neurovascular function. Keywords: Laser Doppler Imaging, vasomotor axon-reflex, capsaicin, Flare response, small fiber neuropathy Financial support: NIH, Langer Family Foundation.
doi:10.1016/j.autneu.2011.05.185
P.171 The quantification of cutaneous autonomic pilomotor innervation C.H. Gibbons, N. Wang, A. Hovaguimian, B.M.W. Illigens, R. Freeman (Beth Israel Deaconess Medical Center, Harvard Medical School Neurology, United States) Background: Our objective in this study is to define the topography of pilomotor nerve fiber density PNFD) in clinically relevant sites, to demonstrate the effectiveness of both dopamine β-hydroxylase (DβH) and tyrosine hydroxylase (TH) to stain for sympathetic adrenergic fibers, and determine the pilomotor nerve fiber density (PNFD) in healthy control subjects and individuals with diabetes using standard light microscopy PGP stained tissue sections. Methods: Thirty-four control and 46 subjects with clinical evidence of diabetic neuropathy were examined and punch skin biopsies at the distal leg, distal thigh, proximal thigh and forearm were obtained. Nerve fibers were labeled with PGP 9.5, TH, DβH, TH and VIP. Images were captured on a confocal microscope in 3 μm optical sections, or labeled by PGP 9.5 for light microscopy sections. Three blinded observers quantified nerve fiber density by counting fibers crossing perpendicular to the axis of the pilomotor muscle. Distribution of nerve fibers by location, subtype and inter-rater reliability was determined. Results: Subjects with neuropathy had reduced pilomotor nerve fiber density (PNFD) compared to controls at the distal leg (69 ± 9 vs 82 ± 16 fibers/mm; p < 0.05), distal thigh (64 ± 14 vs 106 ± 25 fibers/mm; p < 0.01), proximal thigh (85 ± 18 vs. 110 ± 19 fibers/mm; p < 0.05) and forearm (68 ± 12 vs 103 ± 24 fibers/mm; p < 0.01). There was 100% overlap between TH and DβH fibers in arrector pili muscles. The intraclass correlation coefficient was 0.89 (p < 0.001) for interobserver reliability. Conclusion: Quantification of PNFD, using both confocal and light microscopy image analysis techniques, differentiates a group of patients with neuropathy from healthy control subjects. Our results confirm a recently reported method to quantitate PNFD (Nolano
Abstracts / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133
Neurology 2010), expand the technique to light microscopy, use TH and DβH to stain for adrenergic fibers and identify the proximal to distal gradient in diabetic neuropathy. The high interclass correlation coefficients between reviewers confirm that our results are reproducible.
107
Keywords: spinal cord injury, hypertension, plasticity, pain, blood pressure Financial support: Heart and Stroke Foundation of BC and the Yukon (of Canada).
Keywords: Pilomotor, neuropathy, sympathetic, adrenergic, diabetes doi:10.1016/j.autneu.2011.05.187 Financial support: NIH K23NS050209. P.173 Transient changes in BDNF parallel plasticity observed in the IML following distal axon injury doi:10.1016/j.autneu.2011.05.186 A.P. Coulibaly, B.F. Walsh, L.G. Isaacson(Miami University - Zoology, United States) P.172 The role of capsaicin-sensitive sensory neurons in autonomic dysreflexia in the spinal cord injured-rat L.M. Ramer (University of British Columbia -International Collaboration on Repair Discoveries, Canada), A.P. van Stolk (UBC -ICORD, Canada), J.A. Inskip (Simon Fraser University -Biomedical Physiology and Kinesiology, Canada), M.S. Ramer (University of British Columbia International Collaboration on Repair Discoveries, Canada), A.V. Krassioukov (University of British Columbia -Physical Medicine and Rehabilitation, Canada) Background: A subpopulation of sensory neurons express the transient receptor potential cation channel subfamily V member 1 (TRPV1). It is activated by heat (>43 °C), low pH, and capsaicin (the pungent ingredient in hot chili peppers), and has emerged as a promising target for the treatment of pain. Both pain and autonomic dysreflexia that develop in the wake of SCI have been attributed to maladaptive sensory plasticity. We hypothesize that TRPV-1-positive afferents contribute to the development of autonomic dysreflexia (AD). Methods: Wistar rats (300 g) received a complete transection of the spinal cord at the third (T3) or tenth (T10) thoracic segment or sham injury (durotomy without SCI). After 1–12 weeks, dorsal root ganglia (DRGs) and spinal cords were harvested and analyzed immunohistochemically to examine soma size of TRPV1-positive afferents and density of their central projections. A subset of rats received vehicle or capsaicin via intrathecal injection at 28 days; at 30 days, the severity of AD was assessed in these animals, by recording intra-arterial blood pressure during colo-rectal distension (CRD). Results: TRPV-1-positive nociceptors exhibited hypertrophy after T3 SCI. Nociceptor hypertrophy only occurred in DRGs below the level of SCI, and was more pronounced after high thoracic (T3) SCI. Hypertrophy was pronounced in DRGs far distal to SCI, and occurred in DRGs that contained both somatic (L4,L5) and visceral (L6,S1) afferents. TRPV-1-expressing nociceptors also appeared to sprout within the lumbar dorsal horn: the area occupied by TRPV-1-positive axons was increased one month after SCI. CRD-evoked hypertension was less pronounced in rats treated with capsaicin than in vehicletreated animals. Conclusion: TRPV-1-expressing nociceptors respond to SCI by undergoing somal hypertrophy and expanding their central terminals in the lumbar dorsal horn. Selective elimination of the central projections of these nociceptors reduces the severity of AD in rats with SCI. We are now examining the intriguing possibility that spontaneous activity in these neurons may contribute to the development of AD after SCI.
The neurotrophic regulation of sympathetic preganglionic neurons located in the intermediolateral cell column (IML) is poorly understood. We previously reported transient changes in the adult IML following injury to the cervical sympathetic trunk (CST), the distal axons entering the superior cervical ganglion (SCG). At 1 week following CST transection, activated astrocytes and microglia and increased numbers of trkB immunoreactive (− ir) oligodendrocytes populated the IML at levels C8-T1. This glial plasticity was paralleled by a decrease in choline acetyltransferase (ChAT)-ir neurons. Though ChAT was lost, neurons were present and expressed the injury marker activating transcription factor (ATF)-3. At 3 weeks, glial activation was absent, ChAT-ir neurons showed recovery, and ATF3-ir neurons declined to 13% of IML neurons. The number and appearance of ChAT-ir neurons were similar to controls at 10 weeks following injury. The changes in trkB suggested a role for the neurotrophin brain derived neurotrophic factor (BDNF) during this period of plasticity and provided the basis for the present study where we used western blot analysis to investigate BDNF levels in the spinal cord at 1 week, 3 weeks, and 10 weeks following CST transection. At 1 week following injury, when glial cells respond and ChAT is decreased, the 24 kDa isoform showed a significant decrease in all cases and the 37 kDa isoform was decreased in 4 of the 5 cases examined. At 21 days, when gliosis has subsided and ChAT expression is recovering, the 37 kDa form was significantly increased. BDNF returned to control values at 10 weeks following injury. The transient changes in BDNF may play a role in the IML neuroplasticity observed following injury. The BDNF protein in the cord is likely produced locally since the transected axons do not reinnervate the SCG by 10 weeks and could not derive BDNF from their SCG target. Keywords: sympathetic preganglionic neuron, neurotrophin regulation, choline acetyltransferase, trkB Financial support: NIHNS051206 to LGI, Undergraduate Research grant to BFW.
doi:10.1016/j.autneu.2011.05.188
P.174 Spectral analysis of cardiovascular control after spinal cord injury in rats: Effect of time post-injury J.A. Inskip (Simon Fraser University/University of British Columbia Biomedical Physiology and Kinesiology/International Collaboration on Repair Discoveries, Canada), L.M. Ramer (University of British Columbia -International Collaboration on Repair Discoveries/Zoology, Canada), M.S. Ramer (University of British Columbia -International Collaboration on Repair Discoveries/Zoology, Canada), A.V. Krassiou-