ORIGINAL ARTICLE
Spinal cord compression injury in lysophosphatidic acid 1 receptor-null mice promotes maladaptive pronociceptive descending control s1, F.R. de Fonseca3, J. Chun4, M. Suardıaz1, I. Galan-Arriero2,#, G. Avila-Martin2,#, G. Estivill-Torru 5 6 2,7 mez-Soriano , E. Bravo-Esteban , J. Taylor J. Go n Clınica Intercentros de Neurociencias, Instituto de Investigacio n Biome dica de Malaga (IBIMA), Hospitales Universitarios 1 Unidad de Gestio Regional de Malaga y Virgen de la Victoria, Malaga, Spain jicos, Toledo, Spain 2 Sensorimotor Function Group, Hospital Nacional de Paraple n Clınica de Salud Mental, Instituto de Investigacio n Biome dica de Malaga (IBIMA), Hospitales Universitarios Regional de 3 Unidad de Gestio Malaga y Virgen de la Victoria, Malaga, Spain 4 Department of Molecular and Cellular Neuroscience, Dorris Neuroscience Centre The Scripps Research Institute, La Jolla, USA n en Fisioterapia Toledo (GIFTO), E.U.E. Fisioterapia de Toledo, Universidad de Castilla la Mancha, USA 5 Grupo de Investigacio 6 Neurorehabilitation Group, Instituto Cajal, Council for Scientific Research (CSIC), Madrid, Spain 7 Stoke Mandeville Spinal Research, National Spinal Injuries Centre, Aylesbury, UK
Correspondence Julian Taylor E-mail: [email protected], [email protected] #These authors have contributed equally to this study. Funding sources This work has been supported by the follown Mutua Mading funding sources: Fundacio ~a 2013, INNPACTO (Ministerio de rilen n, IPT-010000-2010-016), Ciencia e Innovacio Consorcio ‘Dendria-Draconis Pharma S.L.’ gico (Centro para el Desarrollo Tecnolo Industrial), Instituto de Salud Carlos III PI070806 and PI11/00592, and finally IE05009 to MS (Consejerıa de Sanidad, JCCM). Conflicts of interest The authors have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, their work.
Accepted for publication 6 February 2015 doi:10.1002/ejp.695
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Abstract Background: Although activation of the lysophosphatidic acid receptor 1 (LPA1) is known to mediate pronociceptive effects in peripheral pain models, the role of this receptor in the modulation of spinal nociception following spinal cord injury (SCI) is unknown. Aim: In this study, LPA1 regulation of spinal excitability mediated by supraspinal descending antinociceptive control systems was assessed following SCI in both wild-type (WT) and maLPA1-null receptor mice. Methods: The effect of a T8 spinal compression in WT and maLPA1null mice was assessed up to 1 month after SCI using histological, immunohistochemical and behavioural techniques analysis including electrophysiological recording of noxious toes-Tibialis Anterior (TA) stimulus-response reflex activity. The effect of a T3 paraspinal transcutaneous electrical conditioning stimulus on TA noxious reflex temporal summation was also assessed. Results: Histological analysis demonstrated greater dorsolateral funiculus damage after SCI in maLPA1-null mice, without a change in the stimulus-response function of the TA noxious reflex when compared to WT mice. While T3 conditioning stimulation in the WT group inhibited noxious TA reflex temporal summation after SCI, this stimulus strongly excited TA reflex temporal summation in maLPA1-null mice. The functional switch from descending inhibition to maladaptive facilitation of central excitability of spinal nociception demonstrated in maLPA1-null mice after SCI was unrelated to a general change in reflex activity. Conclusions: These data suggest that the LPA1 receptor is necessary for inhibition of temporal summation of noxious reflex activity, partly mediated via long-tract descending modulatory systems acting at the spinal level.
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What0 s already known about this topic? • Lysophosphatidic acid (LPA) mediates pronociceptive effects in nerve injury models via the LPA1 receptor. • LPA is released following spinal cord injury (SCI). What does this study add?
• maLPA1-null receptor mice show greater dorsolateral funiculus damage after thoracic SCI.
• SCI in maLPA1-null receptor mice leads to maladaptive descending pronociceptive control.
• The LPA1 receptor mediates segmental antinociception partly by maintaining the integrity of long-tract descending inhibitory modulatory systems.
1. Introduction Sensorimotor pathophysiology following spinal cord injury (SCI) includes development of abnormal noxious reflex function which is associated with the spasticity syndrome (Gomez-Soriano et al., 2012; Bravo-Esteban et al., 2014; G omez-Soriano et al., 2015). Myelin breakdown products following SCI include bioactive phospholipids such as lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3-phosphate) (Lukacova et al., 1996) which mediate a variety of physiological functions (Choi et al., 2008), including cell survival (Weiner et al., 1998; Ye et al., 2002a). A family of specific G protein-coupled receptors mediate the effects of LPA (Anliker and Chun, 2004), while the LPA1 receptor subtype has previously been implicated in the regulation of myelination (Weiner et al., 1998). Following peripheral nerve injury LPA initiates neuropathic pain through the activation of the LPA1 receptor subtype (Inoue et al., 2004; Ma et al., 2013). However, the role of the LPA1 receptor in mediating spinal nociception and change in sensory function after central nervous system injury, such as SCI, has not been demonstrated. This study was based on the hypothesis that release of LPA following thoracic spinal compression (Huang et al., 2007; Lim et al., 2013) would specifically facilitate segmental excitability to noxious stimuli, which in turn would be related to the development of neuropathic pain and the spasticity syndrome after SCI (Finnerup et al., 2007; Zhao et al., 2007; Avila-Martin et al., 2011; Gomez-Soriano et al., 2012). The study of temporal summation of the © 2015 European Pain Federation - EFICâ
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toes–Tibialis Anterior (TA) noxious reflex response in response to repeated C-fibre activation following T9 SCI contusion, has shown that descending antinociception and enhanced 5-HT lumbar innervation are promoted following treatment with a novel neurotrophic factor (Avila-Martin et al., 2011). These results suggest that loss of descending antinociception and the development of maladaptive facilitatory control of spinal excitability shown in peripheral nerve injury models (Burgess et al., 2002; Suzuki et al., 2004) may also be characteristic of SCI. The maLPA1-null or ‘Malaga variant’ mouse (Estivill-Torrus et al., 2008) is a stable variant of the original LPA1-null mutant (Contos et al., 2000) that carries the deletion of exon 3, containing the transmembrane domains I–VI of the receptor. Therefore, the canonical Lpar1 knockout and maLPA1-null mice are similar. The maLPA1-null mouse has been used to assess oligodendrocyte differentiation and myelination (Garcia-Diaz et al., 2014). Accordingly, the objective of this study was to examine the role of the LPA1 receptor in mediating spinal excitability to noxious stimuli following SCI in maLPA1-null receptor mice (Estivill-Torrus et al., 2008; Santin et al., 2009), with a specific focus on examining noxious reflex excitability changes mediated by descending antinociceptive modulatory systems (Villanueva et al., 1986; Taylor et al., 1991; Gozariu et al., 1997; Avila-Martin et al., 2011). In contrast to the original hypothesis of this study, the results support an inhibitory role of LPA mediated via the LPA1 receptor subtype in the modulation of segmental nociception below the SCI.
2. Materials and methods The experiments adhered to the European guidelines for animal experiments (http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm), and were approved by the Animal welfare ethical committee of the ‘Hospital Nacional de Parapl ejicos’ in Toledo. Wild-type (WT) and maLPA1-null adult (4–5 weeks) male mice were maintained in the animal resource unit of the Hospital Nacional de Parapl ejicos in Toledo, and provided with food and water ad libitum. The light/dark regime was set from 8:00 a.m. until 20:00 p.m.
2.1 Generation of the maLPA1-null mouse The maLPA1-null mouse was derived spontaneously from the original LPA1-null mouse during colony expansion by crossing heterozygous foundational parEur J Pain 20 (2016) 176--185
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Figure 3 Effect of T3 paraspinal transcutaneous conditioning (Cond) stimulation on nociceptive reflex temporal summation in (A, C) wild-type (WT) and (B, D) maLPA1-null mice 28 days after T8 static spinal compression (SCI). (A, C) T3 conditioning stimulation inhibited noxious toes-TA reflex temporal summation evoked with 1 mA test stimuli in the WT-T8 SCI group, in contrast to (B, D) excitation of noxious reflex activity in the maLPA1-null SCI group mice. Scale bars: (A) 5 lV/10s, (B) 10 lV/10s. Two-way ANOVA: ***p < 0.01. (E) Analysis of the 2nd–8th and 9th–16th reflex response (Resp.) recorded during the temporal summation protocol with 1 mA test stimuli revealed inhibition and excitation of toes-TA reflex activity in the WT and maLPA1-null groups, respectively. (F) In contrast, differential modulation of noxious toes-TA reflex activity evoked with 10 mA test stimuli following T3 conditioning stimulation was identified only during the 9th–16th reflex responses (9–16 Resp.). (C and D) Unpaired t-test: *p < 0.05 **p < 0.01, ***p < 0.01.
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2.4 Static spinal compression histology and immunohistochemical analysis At 28 days after SCI, animals were euthanised with sodium pentobarbital followed by intracardiac perfusionwith saline and a mixture of 4% paraformaldehyde, 75 mM lysine and 10 mM sodium metaperiodate in 0.1 M phosphate buffer. The extracted spinal tissue was post-fixed for 4 h and then transferred to 30% sucrose in 0.1 M phosphate buffer and kept at 4 °C for at least 2 days to provide cryoprotection. To visualise the injured tissue area after SCI injury, 30-lm tissue sections taken from the T7–T9 spinal cord were processed with a microtome (Microm HM450; Microm Laborgerate, S.L. part of Thermofisher Scientific, Barcelona, Spain). Thoracic spinal tissue was prepared with van Gieson’s stain (Avila-Martin et al., 2011). To evaluate serotonin immunohistochemistry within the lumbar spinal cord, 30-lm-thick sections (Leica CM1900; Leica Microsistemas S.L.U., Barcelona, Spain) of the lumbar L4–L5 tissue were processed as previously described (Avila-Martin et al., 2011), using a primary antibody against serotonin (rabbit anti-5HT, 1:4000, Ref: S5545, Sigma-Aldrich Quımica S.L., Madrid, Spain) with a conjugated secondary antibody Alexa Fluor 594 (1:1000; 1 h 4 °C; anti-rabbit, Ref: A11012, Invitrogen, Alcobendas, Madrid, Spain).
2.5 Image analysis Analysis of both the area of the static spinal compression injury and serotonin immunohistochemistry within the dorsal horn of the lumbar spinal cord was calculated as described previously (Avila-Martin et al., 2011). The serotonin-positive area within the dorsal horn was calculated in lm2, averaged and presented as group data (minimum of three images/ section/animal, n = 6–8 mice per group). All analytical anatomical procedures were performed blind, without knowledge of the experimental conditions.
2.6 Statistical analysis Data were analysed using a one-way ANOVA for comparisons between groups, while a two-way ANOVA was used for longitudinal analysis such as with the Rotarod test, or for analysis of noxious toes-TA reflex temporal summation (Graphpad Prism 6.00; GraphPad Software, Inc., La Jolla, CA, USA). Post hoc tests were performed with the Bonferroni test (Rotarod test), the Tukey’s post hoc test (neurophysiological data), and the paired and unpaired Student’s t-test (immunohistochemistry).
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3. Results 3.1 Histological and immunohistochemical analysis following static spinal compression in maLPA1-null mice Analysis of T7–9 spinal sections processed with van Gieson0 s stain (Fig. 1A and B) provided an estimation of the area of injured tissue in WT and maLPA1-null mice 28 days after SCI. The total area of injured tissue was similar when compared between WT and maLPA1-null mice (data not presented). However, a more detailed histological analysis of injured tissue within the dorsal and dorsolateral funiculi revealed greater damage to the latter area in maLPA1-null mice (53 13%, expressed as the percentage of the total injured tissue) compared to the WT group (17 10%) following static spinal compression (Fig. 1C). Immunohistochemical analysis of the serotonin (5HT) area within the lumbar dorsal horn demonstrated a 53% decrease in innervation level following SCI in WT mice compared to sham animals (1827 302 to 850 164, Fig. 1D, *p < 0.05). In contrast, lumbar 5HT innervation levels in maLPA1-null mice were not changed following static spinal compression (1189 165 to 1236 189, Fig. 1D).
3.2 Locomotor function and noxious reflex activity after SCI in maLPA1 null mice To measure locomotor function in animals, the Rotarod test was performed up to 28 days after SCI (Fig. 2A). Both WT and maLPA1-null mice revealed a reduction in motor capacity at day 4 after static spinal compression injury. However, while WT mice progressively recovered locomotor function up to 84 8% of the pre-injury level at 28 days after SCI, maLPA1-null mice demonstrated significantly less recovery (70 8%, ANOVA, p < 0.05 when compared to the WT group). Two-way ANOVA analysis of the noxious toes-TA reflex stimulus-response function (Fig. 2B) revealed significant differences between experimental groups (p < 0.001) and between stimulus intensities (p < 0.001). Specifically, the Bonferroni multiple comparison test detected higher toes-TA noxious reflex activity in response to 10 mA stimulation in the WT-Sham group compared to the WT-SCI (6.0 1.0 mV.ms, p < 0.001), maLPA1-null Sham (4.0 1.8 mV.ms, p < 0.001) and maLPA1-null SCI groups (9.0 1.0 mV.ms, p < 0.001, Fig. 2B). These data suggest that SCI in the WT animals leads to
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Figure 1 Histological and immunohistochemical analysis of T8 static spinal compression injury in wild-type (WT) and maLPA1-null mice at 28 days. (A, B) van Gieson’s staining revealed greater dorsal spinal cord injury (SCI) in the maLPA1-null group (B) compared to the wild-type (WT) group (A) following T8 static compression injury (209). (C) Quantification of the bilateral dorsolateral spinal injury area (see A, B) revealed greater injury area in the maLPA1-null group following T8 compression (Student0 s t-test, *p < 0.05). (D) A reduction in serotoninergic immunoreactivity within the lumbar dorsal horn was identified after T8 SCI in the WT group, but not in the maLPA1-null group (one-way ANOVA, *p < 0.05).
hyporreflexia and that the noxious toes-TA reflex function was not significantly changed in the maLPA1-null animals (Fig. 2B).
3.3 Noxious reflex temporal summation and phasic descending modulation after SCI in maLPA1-null mice Temporal summation of noxious toes-TA reflex activity following T8 SCI was observed in both the WT and maLPA1-null mice when compared to the respective sham-operated groups. Specifically, temporal summation of the 9th–16th reflex response indicated a ‘wind-up’ of noxious reflex activity in the WT-SCI group (110 10 to 208 30%) and maLPA1-null (125 13 to 189 18%) at 28 days after T8 SCI. Application of the T3 paraspinal transcutaneous electrical conditioning stimulus above the SCI strongly inhibited the noxious toes-TA reflex temporal summation in WT mice when compared to reflex activity recorded before the conditioning stimulus 180 Eur J Pain 20 (2016) 176--185
(Fig. 3A and C). This activation of descending inhibitory modulation contrasted to the excitation mediated by the conditioning stimulus in maLPA1-null animals (Fig. 3B and D). Moreover, specific analysis of the 2nd–8th and 9th–16th toes-TA noxious reflex response during temporal summation indicated that the T3 conditioning stimulus in the WT-SCI group inhibited reflex responses evoked with 1.0 mA (Fig 3E), but was only detected during the second phase of temporal summation using the 10 mA test stimuli (Fig 3F). In contrast in the maLPA1-null group, the T3 conditioning stimulus increased noxious toes-TA reflex activity during both the 2nd–8th and 9th–16th stimulus phases of temporal summation with 1.0 mA (Fig. 3E) and 10.0 mA test stimuli after SCI (Fig. 3F). Importantly, no difference was observed for initial TA reflex excitability recorded after the first stimulus before and after SCI in the respective experimental groups, indicating that the modulatory change in the noxious reflex ‘wind-up’ response following the T3 conditioning stimulation
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Figure 2 Recovery of locomotor function and analysis of the stimulus-response function of the noxious toes-TA reflex response in wild-type (WT) and maLPA1-null mice after T8 static compression injury. (A) Locomotor capacity measured with the Rotarod test before and weekly up to 28 days after SCI (two-way ANOVA, *p < 0.05). (B) Noxious toes-TA reflex stimulus-response function in WT and maLPA1-null mice 28 days following T8 static spinal compression (two-way ANOVA and Bonferroni-adjusted comparison, ***p < 0.01).
was unrelated to a general change in reflex stimulus-response excitability (see Fig. 2).
4. Discussion This study demonstrates that the maLPA1 receptor subtype is essential in maintaining inhibition of noxious reflex excitability following SCI (c.f. Inoue et al., 2004; Ma et al., 2013). Greater damage to the dorsal and dorsolateral funiculi identified after SCI in maLPA1-null mice suggests that this receptor plays a central role in maintaining the integrity of long-tract pathways (Weiner et al., 1998; Ye et al., 2002b; Matsushita et al., 2005; Nogaroli et al., 2009; Anliker et al., 2013). The functional switch from descending inhibition to excitation of spinal noxious reflex activity observed following paraspinal transcutaneous conditioning stimulation applied above the injury site, is presumably mediated via non-serotoninergic pathways (Fleetwood-Walker et al., 1988; Taylor et al., 1991), which may play an important role in the development of maladaptive descending pronociceptive modulation after SCI (Villanueva et al., 1986; Gozariu et al., 1997).
4.1 Thoracic spinal cord compression in maLPA1-null mice In this study, a T8 spinal cord compression injury model, rather than a moderate contusion injury model was used (Huang et al., 2007). Static spinal compression in the mouse leads to a 84% loss of oligodendrocytes within the dorsal column at 28 days after SCI (Lim et al., 2013). In this study, extensive damage to the dorsal funiculus was identified in © 2015 European Pain Federation - EFICâ
maLPA1-null compared to WT mice, suggesting that the LPA1 receptor mediates a neuroprotective role following SCI (Weiner et al., 1998; Ye et al., 2002b), possibly through the protection of oligodendrocytes (Weiner et al., 1998). This hypothesis is supported partly by loss of locomotor function recovery observed up to 28 days following SCI in maLPA1null mice. Although serotonin-positive density was lower in maLPA1-null compared to WT mice (c.f. Harrison et al., 2003), no change in 5-HT-positive density levels were identified within the lumbar cord following SCI. Taken together, these results suggest that central excitability of noxious toes-TA reflex activity is modulated by non-serotoninergic descending modulatory pathways via the dorsolateral funiculus.
4.2 LPA1 receptor activation does not lead to cutaneous hyperreflexia to noxious stimuli after SCI This study suggests that LPA1 receptor activation following static spinal compression is not pronociceptive, which contrasts with evidence obtained from peripheral nerve injury models (Inoue et al., 2004; Ma et al., 2013). Therefore, the role of the LPA1 receptor in the SCI model may reflect both the neurological level and the grade of the specific CNS injury, where a more severe thoracic lesion would be expected to induce a greater change in spinal reflex excitability. However, the stimulus-response function of the toes-TA noxious reflex measured before SCI in maLPA1-null mice was significantly smaller than that observed in WT mice. This suggests that the level of basal noxious reflex excitability in Eur J Pain 20 (2016) 176--185
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Figure 3 Effect of T3 paraspinal transcutaneous conditioning (Cond) stimulation on nociceptive reflex temporal summation in (A, C) wild-type (WT) and (B, D) maLPA1-null mice 28 days after T8 static spinal compression (SCI). (A, C) T3 conditioning stimulation inhibited noxious toes-TA reflex temporal summation evoked with 1 mA test stimuli in the WT-T8 SCI group, in contrast to (B, D) excitation of noxious reflex activity in the maLPA1-null SCI group mice. Scale bars: (A) 5 lV/10s, (B) 10 lV/10s. Two-way ANOVA: ***p < 0.01. (E) Analysis of the 2nd–8th and 9th–16th reflex response (Resp.) recorded during the temporal summation protocol with 1 mA test stimuli revealed inhibition and excitation of toes-TA reflex activity in the WT and maLPA1-null groups, respectively. (F) In contrast, differential modulation of noxious toes-TA reflex activity evoked with 10 mA test stimuli following T3 conditioning stimulation was identified only during the 9th–16th reflex responses (9–16 Resp.). (C and D) Unpaired t-test: *p < 0.05 **p < 0.01, ***p < 0.01.
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maLPA1-null mice is reduced, which in turn may reflect a general change in 5-HT level and associated control mechanisms (Harrison et al., 2003). Whether reduced development of descending facilitatory modulatory mechanisms plays a role in the low level of noxious spinal reflex activity observed in maLPA1null mice needs to be addressed.
4.3 LPA1 receptor activation mediates phasic descending inhibition of noxious reflex temporal summation after SCI We have previously demonstrated that treatment of T9 contusion SCI with a novel neurotrophic factor mediates de novo descending antinociception of the noxious toes-Tibialis Anterior (TA) reflex activity, in parallel with an increase in 5-HT lumbar innervation (Avila-Martin et al., 2011). In this study, a similar paraspinal transcutaneous conditioning stimulus protocol was instrumental in characterizing descending modulation of noxious reflex excitability in the mouse. Hence, sparing of the dorsolateral funiculus following SCI in WT mice was associated with phasic descending inhibition of noxious toes-TA reflex activity following paraspinal conditioning stimulation. In contrast, a strong phasic descending facilitation of noxious reflex activity was induced in maLPA1-null mice following SCI, suggesting a role for this receptor subtype in the protection of oligodendrocytes (Weiner et al., 1998; Ye et al., 2002b) and the integrity of descending modulatory pathways within the dorsolateral funiculus (Villanueva et al., 1986; Taylor et al., 1991, 1997; Ossipov et al., 2000). This is the first study to demonstrate descending pronociception of noxious reflex activity across the experimental SCI site. Descending modulation of spinal excitability has been demonstrated in several studies, including animal models of neuropathic pain (Suzuki et al., 2004; Vera-Portocarrero et al., 2006; De Felice et al., 2011), while descending pronociceptive control has also been described following peripheral nerve injury (Burgess et al., 2002).
4.4 Methodological advances and limitations in the assessment of spinal and supraspinal modulation of noxious reflex activity following SCI in the mouse The clear demonstration of temporal summation of noxious reflex activity following spinal compression injury in the mouse, compared to the moderate contusion injury model in the rat (Avila-Martin et al., 2011), represents an important methodological
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advance. Although temporal summation of reflex activity has been measured before in the mouse (De Felipe et al., 1998), quantification of this mechanism has been limited in this species due to the fatigability of the reflex response observed during the recording protocol. The improved protocol developed in this study demonstrates that static spinal compression leads to an increase in temporal summation of noxious reflex activity, warranting further investigation into the effect of SCI compression on spinal nociception. It is important to note that classical central sensitization was not evaluated in this study (Woolf, 1996; Li et al., 1999; Herrero et al., 2000), which would have been evident by analysing noxious reflex activity after the temporal summation protocol. However, the pronociceptive effect of the T3 spinal conditioning stimulus on prolonged noxious toes-TA reflex activity observed in maLPA1-null mice after SCI following repeated C-fibre stimulation (see Fig. 3B and D) suggests that central sensitization to noxious input is present after compression SCI. The functional switch observed from descending inhibition to facilitation of noxious spinal reflex excitability following thoracic dorsolateral funiculus damage suggests that this mouse model of SCI should be instrumental in revealing both spinal and supraspinal pathophysiological mechanisms related to the development of the spasticity syndrome and local mechanisms of nociception, which are prevalent following SCI (Avila-Martin et al., 2011). However it is not possible in this study to exclude the contribution of intraspinal control systems organized within the low thoracic or upper lumbar spinal level to greater temporal summation of noxious reflex activity after SCI following activation of descending modulatory pathways (Cadden et al., 1983; Cavallari and Pettersson, 1989; Magnuson et al., 1999). Further examination of the balance and integrity of competing descending inhibitory and facilitatory modulatory pain pathways from the brainstem should be assessed as important control mechanisms of spinal reflex excitability following SCI (Gozariu et al., 1997). In this regard, in direct assessment of the role of descending intraspinal modulatory pathways activated either by paraspinal transcutaneous conditioning stimulation in the rat (Avila-Martin et al., 2011) or by non-invasive techniques in man (Albu et al., 2013) may provide important methodological and mechanistic information regarding the state of these modulatory mechanisms following SCI.
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4.5 LPA1 modulation of the immune system after SCI A confounding factor that could account for the functional effects of the compression SCI in the maLPA1-null mice is the possible effect of LPA1 deficiency on the immune component activated after injury. Activated T-cells are known to express LPA1 and this mechanism is able to regulate their proliferation (Huang et al., 2002). Moreover, LPA1 KO mice are resistant to arthritic disease induction which results from a block of immune cell infiltration (Miyabe et al., 2013). Since T-cells play an important role following traumatic SCI, particularly in modulating inflammation, neurodegeneration and repair (Ankeny and Popovich, 2009), the maladaptive pronociceptive response observed in the maLPA1-null mice in this study may also reflect a blunting of repair processes due to disruption of lymphocyte activation following injury. Future studies of the role of LPA receptors in modulation of sensorimotor function following SCI should also assess the role of T-cell activation as a possible contributing factor.
5. Conclusion Maintenance of long-tract descending antinociceptive modulatory pathways is mediated by the LPA1 receptor following thoracic SCI, which plays a role in the control of phasic inhibition of spinal noxious reflex activity and locomotor recovery. Therefore, this study suggests that the LPA1 receptor plays an important role in neuroprotection of descending modulatory pathways following SCI, possibly by maintaining or inducing remyelination of neuronal long-tract systems across the injury site (Weiner et al., 1998; Ye et al., 2002b; Matsushita et al., 2005; Nogaroli et al., 2009; Anliker et al., 2013). Author contributions All authors were responsible for: (1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be published.
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N., Nanki, T. (2013). Necessity of lysophosphatidic acid receptor 1 for development of arthritis. Arthritis Rheum 65, 2037–2047. Nogaroli, L., Yuelling, L.M., Dennis, J., Gorse, K., Payne, S.G., Fuss, B. (2009). Lysophosphatidic acid can support the formation of membranous structures and an increase in MBP mRNA levels in differentiating oligodendrocytes. Neurochem Res 34, 182–193. Ossipov, M.H., Hong Sun, T., Malan, P. Jr, Lai, J., Porreca, F. (2000). Mediation of spinal nerve injury induced tactile allodynia by descending facilitatory pathways in the dorsolateral funiculus in rats. Neurosci Lett 290, 129–132. Santin, L.J., Bilbao, A., Pedraza, C., Matas-Rico, E., Lopez-Barroso, D., Castilla-Ortega, E., Sanchez-Lopez, J., Riquelme, R., Varela-Nieto, I., de la Villa, P., Suardiaz, M., Chun, J., De Fonseca, F.R., EstivillTorrus, G. (2009). Behavioral phenotype of maLPA1-null mice: Increased anxiety-like behavior and spatial memory deficits. Genes Brain Behav 8, 772–784. Suzuki, R., Rahman, W., Hunt, S.P., Dickenson, A.H. (2004). Descending facilitatory control of mechanically evoked responses is enhanced in deep dorsal horn neurones following peripheral nerve injury. Brain Res 1019, 68–76. Taylor, J.S., Neal, R.I., Harris, J., Ford, T.W., Clarke, R.W. (1991). Prolonged inhibition of a spinal reflex after intense stimulation of distant peripheral nerves in the decerebrated rabbit. J Physiol 437, 71– 83. Taylor, J.S., Friedman, R.F., Munson, J.B., Vierck, C.J. Jr (1997). Stretch hyperreflexia of triceps surae muscles in the conscious cat after dorsolateral spinal lesions. J Neurosci 17, 5004–5015. Vera-Portocarrero, L.P., Zhang, E.T., Ossipov, M.H., Xie, J.Y., King, T., Lai, J., Porreca, F. (2006). Descending facilitation from the rostral ventromedial medulla maintains nerve injury-induced central sensitization. Neuroscience 140, 1311–1320. Villanueva, L., Chitour, D., Le Bars, D. (1986). Involvement of the dorsolateral funiculus in the descending spinal projections responsible for diffuse noxious inhibitory controls in the rat. J Neurophysiol 56, 1185–1195. Weiner, J.A., Hecht, J.H., Chun, J. (1998). Lysophosphatidic acid receptor gene vzg-1/lpA1/edg-2 is expressed by mature oligodendrocytes during myelination in the postnatal murine brain. J Comp Neurol 398, 587–598. Woolf, C.J. (1996). Windup and central sensitization are not equivalent. Pain 66, 105–108. Ye, X., Fukushima, N., Kingsbury, M.A., Chun, J. (2002a). Lysophosphatidic acid in neural signaling. NeuroReport 13, 2169–2175. Ye, X., Ishii, I., Kingsbury, M.A., Chun, J. (2002b). Lysophosphatidic acid as a novel cell survival/apoptotic factor. Biochim Biophys Acta 1585, 108–113. Zhao, P., Waxman, S.G., Hains, B.C. (2007). Modulation of thalamic nociceptive processing after spinal cord injury through remote activation of thalamic microglia by cysteine cysteine chemokine ligand 21. J Neurosci 27, 8893–8902.
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Spinal cord compression injury in lysophosphatidic acid 1 receptor-null mice promotes maladaptive pronociceptive descending control s1, F.R. de Fonseca3, J. Chun4, M. Suardıaz1, I. Galan-Arriero2,#, G. Avila-Martin2,#, G. Estivill-Torru 5 6 2,7 mez-Soriano , E. Bravo-Esteban , J. Taylor J. Go n Clınica Intercentros de Neurociencias, Instituto de Investigacio n Biome dica de Malaga (IBIMA), Hospitales Universitarios 1 Unidad de Gestio Regional de Malaga y Virgen de la Victoria, Malaga, Spain jicos, Toledo, Spain 2 Sensorimotor Function Group, Hospital Nacional de Paraple n Clınica de Salud Mental, Instituto de Investigacio n Biome dica de Malaga (IBIMA), Hospitales Universitarios Regional de 3 Unidad de Gestio Malaga y Virgen de la Victoria, Malaga, Spain 4 Department of Molecular and Cellular Neuroscience, Dorris Neuroscience Centre The Scripps Research Institute, La Jolla, USA n en Fisioterapia Toledo (GIFTO), E.U.E. Fisioterapia de Toledo, Universidad de Castilla la Mancha, USA 5 Grupo de Investigacio 6 Neurorehabilitation Group, Instituto Cajal, Council for Scientific Research (CSIC), Madrid, Spain 7 Stoke Mandeville Spinal Research, National Spinal Injuries Centre, Aylesbury, UK
Correspondence Julian Taylor E-mail: [email protected], [email protected] #These authors have contributed equally to this study. Funding sources This work has been supported by the follown Mutua Mading funding sources: Fundacio ~a 2013, INNPACTO (Ministerio de rilen n, IPT-010000-2010-016), Ciencia e Innovacio Consorcio ‘Dendria-Draconis Pharma S.L.’ gico (Centro para el Desarrollo Tecnolo Industrial), Instituto de Salud Carlos III PI070806 and PI11/00592, and finally IE05009 to MS (Consejerıa de Sanidad, JCCM). Conflicts of interest The authors have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence, their work.
Accepted for publication 6 February 2015 doi:10.1002/ejp.695
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Abstract Background: Although activation of the lysophosphatidic acid receptor 1 (LPA1) is known to mediate pronociceptive effects in peripheral pain models, the role of this receptor in the modulation of spinal nociception following spinal cord injury (SCI) is unknown. Aim: In this study, LPA1 regulation of spinal excitability mediated by supraspinal descending antinociceptive control systems was assessed following SCI in both wild-type (WT) and maLPA1-null receptor mice. Methods: The effect of a T8 spinal compression in WT and maLPA1null mice was assessed up to 1 month after SCI using histological, immunohistochemical and behavioural techniques analysis including electrophysiological recording of noxious toes-Tibialis Anterior (TA) stimulus-response reflex activity. The effect of a T3 paraspinal transcutaneous electrical conditioning stimulus on TA noxious reflex temporal summation was also assessed. Results: Histological analysis demonstrated greater dorsolateral funiculus damage after SCI in maLPA1-null mice, without a change in the stimulus-response function of the TA noxious reflex when compared to WT mice. While T3 conditioning stimulation in the WT group inhibited noxious TA reflex temporal summation after SCI, this stimulus strongly excited TA reflex temporal summation in maLPA1-null mice. The functional switch from descending inhibition to maladaptive facilitation of central excitability of spinal nociception demonstrated in maLPA1-null mice after SCI was unrelated to a general change in reflex activity. Conclusions: These data suggest that the LPA1 receptor is necessary for inhibition of temporal summation of noxious reflex activity, partly mediated via long-tract descending modulatory systems acting at the spinal level.
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What0 s already known about this topic? • Lysophosphatidic acid (LPA) mediates pronociceptive effects in nerve injury models via the LPA1 receptor. • LPA is released following spinal cord injury (SCI). What does this study add?
• maLPA1-null receptor mice show greater dorsolateral funiculus damage after thoracic SCI.
• SCI in maLPA1-null receptor mice leads to maladaptive descending pronociceptive control.
• The LPA1 receptor mediates segmental antinociception partly by maintaining the integrity of long-tract descending inhibitory modulatory systems.
1. Introduction Sensorimotor pathophysiology following spinal cord injury (SCI) includes development of abnormal noxious reflex function which is associated with the spasticity syndrome (Gomez-Soriano et al., 2012; Bravo-Esteban et al., 2014; G omez-Soriano et al., 2015). Myelin breakdown products following SCI include bioactive phospholipids such as lysophosphatidic acid (LPA, 1-acyl-2-sn-glycerol-3-phosphate) (Lukacova et al., 1996) which mediate a variety of physiological functions (Choi et al., 2008), including cell survival (Weiner et al., 1998; Ye et al., 2002a). A family of specific G protein-coupled receptors mediate the effects of LPA (Anliker and Chun, 2004), while the LPA1 receptor subtype has previously been implicated in the regulation of myelination (Weiner et al., 1998). Following peripheral nerve injury LPA initiates neuropathic pain through the activation of the LPA1 receptor subtype (Inoue et al., 2004; Ma et al., 2013). However, the role of the LPA1 receptor in mediating spinal nociception and change in sensory function after central nervous system injury, such as SCI, has not been demonstrated. This study was based on the hypothesis that release of LPA following thoracic spinal compression (Huang et al., 2007; Lim et al., 2013) would specifically facilitate segmental excitability to noxious stimuli, which in turn would be related to the development of neuropathic pain and the spasticity syndrome after SCI (Finnerup et al., 2007; Zhao et al., 2007; Avila-Martin et al., 2011; Gomez-Soriano et al., 2012). The study of temporal summation of the © 2015 European Pain Federation - EFICâ
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toes–Tibialis Anterior (TA) noxious reflex response in response to repeated C-fibre activation following T9 SCI contusion, has shown that descending antinociception and enhanced 5-HT lumbar innervation are promoted following treatment with a novel neurotrophic factor (Avila-Martin et al., 2011). These results suggest that loss of descending antinociception and the development of maladaptive facilitatory control of spinal excitability shown in peripheral nerve injury models (Burgess et al., 2002; Suzuki et al., 2004) may also be characteristic of SCI. The maLPA1-null or ‘Malaga variant’ mouse (Estivill-Torrus et al., 2008) is a stable variant of the original LPA1-null mutant (Contos et al., 2000) that carries the deletion of exon 3, containing the transmembrane domains I–VI of the receptor. Therefore, the canonical Lpar1 knockout and maLPA1-null mice are similar. The maLPA1-null mouse has been used to assess oligodendrocyte differentiation and myelination (Garcia-Diaz et al., 2014). Accordingly, the objective of this study was to examine the role of the LPA1 receptor in mediating spinal excitability to noxious stimuli following SCI in maLPA1-null receptor mice (Estivill-Torrus et al., 2008; Santin et al., 2009), with a specific focus on examining noxious reflex excitability changes mediated by descending antinociceptive modulatory systems (Villanueva et al., 1986; Taylor et al., 1991; Gozariu et al., 1997; Avila-Martin et al., 2011). In contrast to the original hypothesis of this study, the results support an inhibitory role of LPA mediated via the LPA1 receptor subtype in the modulation of segmental nociception below the SCI.
2. Materials and methods The experiments adhered to the European guidelines for animal experiments (http://ec.europa.eu/environment/chemicals/lab_animals/legislation_en.htm), and were approved by the Animal welfare ethical committee of the ‘Hospital Nacional de Parapl ejicos’ in Toledo. Wild-type (WT) and maLPA1-null adult (4–5 weeks) male mice were maintained in the animal resource unit of the Hospital Nacional de Parapl ejicos in Toledo, and provided with food and water ad libitum. The light/dark regime was set from 8:00 a.m. until 20:00 p.m.
2.1 Generation of the maLPA1-null mouse The maLPA1-null mouse was derived spontaneously from the original LPA1-null mouse during colony expansion by crossing heterozygous foundational parEur J Pain 20 (2016) 176--185
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Figure 3 Effect of T3 paraspinal transcutaneous conditioning (Cond) stimulation on nociceptive reflex temporal summation in (A, C) wild-type (WT) and (B, D) maLPA1-null mice 28 days after T8 static spinal compression (SCI). (A, C) T3 conditioning stimulation inhibited noxious toes-TA reflex temporal summation evoked with 1 mA test stimuli in the WT-T8 SCI group, in contrast to (B, D) excitation of noxious reflex activity in the maLPA1-null SCI group mice. Scale bars: (A) 5 lV/10s, (B) 10 lV/10s. Two-way ANOVA: ***p < 0.01. (E) Analysis of the 2nd–8th and 9th–16th reflex response (Resp.) recorded during the temporal summation protocol with 1 mA test stimuli revealed inhibition and excitation of toes-TA reflex activity in the WT and maLPA1-null groups, respectively. (F) In contrast, differential modulation of noxious toes-TA reflex activity evoked with 10 mA test stimuli following T3 conditioning stimulation was identified only during the 9th–16th reflex responses (9–16 Resp.). (C and D) Unpaired t-test: *p < 0.05 **p < 0.01, ***p < 0.01.
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2.4 Static spinal compression histology and immunohistochemical analysis At 28 days after SCI, animals were euthanised with sodium pentobarbital followed by intracardiac perfusionwith saline and a mixture of 4% paraformaldehyde, 75 mM lysine and 10 mM sodium metaperiodate in 0.1 M phosphate buffer. The extracted spinal tissue was post-fixed for 4 h and then transferred to 30% sucrose in 0.1 M phosphate buffer and kept at 4 °C for at least 2 days to provide cryoprotection. To visualise the injured tissue area after SCI injury, 30-lm tissue sections taken from the T7–T9 spinal cord were processed with a microtome (Microm HM450; Microm Laborgerate, S.L. part of Thermofisher Scientific, Barcelona, Spain). Thoracic spinal tissue was prepared with van Gieson’s stain (Avila-Martin et al., 2011). To evaluate serotonin immunohistochemistry within the lumbar spinal cord, 30-lm-thick sections (Leica CM1900; Leica Microsistemas S.L.U., Barcelona, Spain) of the lumbar L4–L5 tissue were processed as previously described (Avila-Martin et al., 2011), using a primary antibody against serotonin (rabbit anti-5HT, 1:4000, Ref: S5545, Sigma-Aldrich Quımica S.L., Madrid, Spain) with a conjugated secondary antibody Alexa Fluor 594 (1:1000; 1 h 4 °C; anti-rabbit, Ref: A11012, Invitrogen, Alcobendas, Madrid, Spain).
2.5 Image analysis Analysis of both the area of the static spinal compression injury and serotonin immunohistochemistry within the dorsal horn of the lumbar spinal cord was calculated as described previously (Avila-Martin et al., 2011). The serotonin-positive area within the dorsal horn was calculated in lm2, averaged and presented as group data (minimum of three images/ section/animal, n = 6–8 mice per group). All analytical anatomical procedures were performed blind, without knowledge of the experimental conditions.
2.6 Statistical analysis Data were analysed using a one-way ANOVA for comparisons between groups, while a two-way ANOVA was used for longitudinal analysis such as with the Rotarod test, or for analysis of noxious toes-TA reflex temporal summation (Graphpad Prism 6.00; GraphPad Software, Inc., La Jolla, CA, USA). Post hoc tests were performed with the Bonferroni test (Rotarod test), the Tukey’s post hoc test (neurophysiological data), and the paired and unpaired Student’s t-test (immunohistochemistry).
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3. Results 3.1 Histological and immunohistochemical analysis following static spinal compression in maLPA1-null mice Analysis of T7–9 spinal sections processed with van Gieson0 s stain (Fig. 1A and B) provided an estimation of the area of injured tissue in WT and maLPA1-null mice 28 days after SCI. The total area of injured tissue was similar when compared between WT and maLPA1-null mice (data not presented). However, a more detailed histological analysis of injured tissue within the dorsal and dorsolateral funiculi revealed greater damage to the latter area in maLPA1-null mice (53 13%, expressed as the percentage of the total injured tissue) compared to the WT group (17 10%) following static spinal compression (Fig. 1C). Immunohistochemical analysis of the serotonin (5HT) area within the lumbar dorsal horn demonstrated a 53% decrease in innervation level following SCI in WT mice compared to sham animals (1827 302 to 850 164, Fig. 1D, *p < 0.05). In contrast, lumbar 5HT innervation levels in maLPA1-null mice were not changed following static spinal compression (1189 165 to 1236 189, Fig. 1D).
3.2 Locomotor function and noxious reflex activity after SCI in maLPA1 null mice To measure locomotor function in animals, the Rotarod test was performed up to 28 days after SCI (Fig. 2A). Both WT and maLPA1-null mice revealed a reduction in motor capacity at day 4 after static spinal compression injury. However, while WT mice progressively recovered locomotor function up to 84 8% of the pre-injury level at 28 days after SCI, maLPA1-null mice demonstrated significantly less recovery (70 8%, ANOVA, p < 0.05 when compared to the WT group). Two-way ANOVA analysis of the noxious toes-TA reflex stimulus-response function (Fig. 2B) revealed significant differences between experimental groups (p < 0.001) and between stimulus intensities (p < 0.001). Specifically, the Bonferroni multiple comparison test detected higher toes-TA noxious reflex activity in response to 10 mA stimulation in the WT-Sham group compared to the WT-SCI (6.0 1.0 mV.ms, p < 0.001), maLPA1-null Sham (4.0 1.8 mV.ms, p < 0.001) and maLPA1-null SCI groups (9.0 1.0 mV.ms, p < 0.001, Fig. 2B). These data suggest that SCI in the WT animals leads to
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Figure 1 Histological and immunohistochemical analysis of T8 static spinal compression injury in wild-type (WT) and maLPA1-null mice at 28 days. (A, B) van Gieson’s staining revealed greater dorsal spinal cord injury (SCI) in the maLPA1-null group (B) compared to the wild-type (WT) group (A) following T8 static compression injury (209). (C) Quantification of the bilateral dorsolateral spinal injury area (see A, B) revealed greater injury area in the maLPA1-null group following T8 compression (Student0 s t-test, *p < 0.05). (D) A reduction in serotoninergic immunoreactivity within the lumbar dorsal horn was identified after T8 SCI in the WT group, but not in the maLPA1-null group (one-way ANOVA, *p < 0.05).
hyporreflexia and that the noxious toes-TA reflex function was not significantly changed in the maLPA1-null animals (Fig. 2B).
3.3 Noxious reflex temporal summation and phasic descending modulation after SCI in maLPA1-null mice Temporal summation of noxious toes-TA reflex activity following T8 SCI was observed in both the WT and maLPA1-null mice when compared to the respective sham-operated groups. Specifically, temporal summation of the 9th–16th reflex response indicated a ‘wind-up’ of noxious reflex activity in the WT-SCI group (110 10 to 208 30%) and maLPA1-null (125 13 to 189 18%) at 28 days after T8 SCI. Application of the T3 paraspinal transcutaneous electrical conditioning stimulus above the SCI strongly inhibited the noxious toes-TA reflex temporal summation in WT mice when compared to reflex activity recorded before the conditioning stimulus 180 Eur J Pain 20 (2016) 176--185
(Fig. 3A and C). This activation of descending inhibitory modulation contrasted to the excitation mediated by the conditioning stimulus in maLPA1-null animals (Fig. 3B and D). Moreover, specific analysis of the 2nd–8th and 9th–16th toes-TA noxious reflex response during temporal summation indicated that the T3 conditioning stimulus in the WT-SCI group inhibited reflex responses evoked with 1.0 mA (Fig 3E), but was only detected during the second phase of temporal summation using the 10 mA test stimuli (Fig 3F). In contrast in the maLPA1-null group, the T3 conditioning stimulus increased noxious toes-TA reflex activity during both the 2nd–8th and 9th–16th stimulus phases of temporal summation with 1.0 mA (Fig. 3E) and 10.0 mA test stimuli after SCI (Fig. 3F). Importantly, no difference was observed for initial TA reflex excitability recorded after the first stimulus before and after SCI in the respective experimental groups, indicating that the modulatory change in the noxious reflex ‘wind-up’ response following the T3 conditioning stimulation
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Figure 2 Recovery of locomotor function and analysis of the stimulus-response function of the noxious toes-TA reflex response in wild-type (WT) and maLPA1-null mice after T8 static compression injury. (A) Locomotor capacity measured with the Rotarod test before and weekly up to 28 days after SCI (two-way ANOVA, *p < 0.05). (B) Noxious toes-TA reflex stimulus-response function in WT and maLPA1-null mice 28 days following T8 static spinal compression (two-way ANOVA and Bonferroni-adjusted comparison, ***p < 0.01).
was unrelated to a general change in reflex stimulus-response excitability (see Fig. 2).
4. Discussion This study demonstrates that the maLPA1 receptor subtype is essential in maintaining inhibition of noxious reflex excitability following SCI (c.f. Inoue et al., 2004; Ma et al., 2013). Greater damage to the dorsal and dorsolateral funiculi identified after SCI in maLPA1-null mice suggests that this receptor plays a central role in maintaining the integrity of long-tract pathways (Weiner et al., 1998; Ye et al., 2002b; Matsushita et al., 2005; Nogaroli et al., 2009; Anliker et al., 2013). The functional switch from descending inhibition to excitation of spinal noxious reflex activity observed following paraspinal transcutaneous conditioning stimulation applied above the injury site, is presumably mediated via non-serotoninergic pathways (Fleetwood-Walker et al., 1988; Taylor et al., 1991), which may play an important role in the development of maladaptive descending pronociceptive modulation after SCI (Villanueva et al., 1986; Gozariu et al., 1997).
4.1 Thoracic spinal cord compression in maLPA1-null mice In this study, a T8 spinal cord compression injury model, rather than a moderate contusion injury model was used (Huang et al., 2007). Static spinal compression in the mouse leads to a 84% loss of oligodendrocytes within the dorsal column at 28 days after SCI (Lim et al., 2013). In this study, extensive damage to the dorsal funiculus was identified in © 2015 European Pain Federation - EFICâ
maLPA1-null compared to WT mice, suggesting that the LPA1 receptor mediates a neuroprotective role following SCI (Weiner et al., 1998; Ye et al., 2002b), possibly through the protection of oligodendrocytes (Weiner et al., 1998). This hypothesis is supported partly by loss of locomotor function recovery observed up to 28 days following SCI in maLPA1null mice. Although serotonin-positive density was lower in maLPA1-null compared to WT mice (c.f. Harrison et al., 2003), no change in 5-HT-positive density levels were identified within the lumbar cord following SCI. Taken together, these results suggest that central excitability of noxious toes-TA reflex activity is modulated by non-serotoninergic descending modulatory pathways via the dorsolateral funiculus.
4.2 LPA1 receptor activation does not lead to cutaneous hyperreflexia to noxious stimuli after SCI This study suggests that LPA1 receptor activation following static spinal compression is not pronociceptive, which contrasts with evidence obtained from peripheral nerve injury models (Inoue et al., 2004; Ma et al., 2013). Therefore, the role of the LPA1 receptor in the SCI model may reflect both the neurological level and the grade of the specific CNS injury, where a more severe thoracic lesion would be expected to induce a greater change in spinal reflex excitability. However, the stimulus-response function of the toes-TA noxious reflex measured before SCI in maLPA1-null mice was significantly smaller than that observed in WT mice. This suggests that the level of basal noxious reflex excitability in Eur J Pain 20 (2016) 176--185
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A
B
C
D
E
F
Figure 3 Effect of T3 paraspinal transcutaneous conditioning (Cond) stimulation on nociceptive reflex temporal summation in (A, C) wild-type (WT) and (B, D) maLPA1-null mice 28 days after T8 static spinal compression (SCI). (A, C) T3 conditioning stimulation inhibited noxious toes-TA reflex temporal summation evoked with 1 mA test stimuli in the WT-T8 SCI group, in contrast to (B, D) excitation of noxious reflex activity in the maLPA1-null SCI group mice. Scale bars: (A) 5 lV/10s, (B) 10 lV/10s. Two-way ANOVA: ***p < 0.01. (E) Analysis of the 2nd–8th and 9th–16th reflex response (Resp.) recorded during the temporal summation protocol with 1 mA test stimuli revealed inhibition and excitation of toes-TA reflex activity in the WT and maLPA1-null groups, respectively. (F) In contrast, differential modulation of noxious toes-TA reflex activity evoked with 10 mA test stimuli following T3 conditioning stimulation was identified only during the 9th–16th reflex responses (9–16 Resp.). (C and D) Unpaired t-test: *p < 0.05 **p < 0.01, ***p < 0.01.
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maLPA1-null mice is reduced, which in turn may reflect a general change in 5-HT level and associated control mechanisms (Harrison et al., 2003). Whether reduced development of descending facilitatory modulatory mechanisms plays a role in the low level of noxious spinal reflex activity observed in maLPA1null mice needs to be addressed.
4.3 LPA1 receptor activation mediates phasic descending inhibition of noxious reflex temporal summation after SCI We have previously demonstrated that treatment of T9 contusion SCI with a novel neurotrophic factor mediates de novo descending antinociception of the noxious toes-Tibialis Anterior (TA) reflex activity, in parallel with an increase in 5-HT lumbar innervation (Avila-Martin et al., 2011). In this study, a similar paraspinal transcutaneous conditioning stimulus protocol was instrumental in characterizing descending modulation of noxious reflex excitability in the mouse. Hence, sparing of the dorsolateral funiculus following SCI in WT mice was associated with phasic descending inhibition of noxious toes-TA reflex activity following paraspinal conditioning stimulation. In contrast, a strong phasic descending facilitation of noxious reflex activity was induced in maLPA1-null mice following SCI, suggesting a role for this receptor subtype in the protection of oligodendrocytes (Weiner et al., 1998; Ye et al., 2002b) and the integrity of descending modulatory pathways within the dorsolateral funiculus (Villanueva et al., 1986; Taylor et al., 1991, 1997; Ossipov et al., 2000). This is the first study to demonstrate descending pronociception of noxious reflex activity across the experimental SCI site. Descending modulation of spinal excitability has been demonstrated in several studies, including animal models of neuropathic pain (Suzuki et al., 2004; Vera-Portocarrero et al., 2006; De Felice et al., 2011), while descending pronociceptive control has also been described following peripheral nerve injury (Burgess et al., 2002).
4.4 Methodological advances and limitations in the assessment of spinal and supraspinal modulation of noxious reflex activity following SCI in the mouse The clear demonstration of temporal summation of noxious reflex activity following spinal compression injury in the mouse, compared to the moderate contusion injury model in the rat (Avila-Martin et al., 2011), represents an important methodological
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advance. Although temporal summation of reflex activity has been measured before in the mouse (De Felipe et al., 1998), quantification of this mechanism has been limited in this species due to the fatigability of the reflex response observed during the recording protocol. The improved protocol developed in this study demonstrates that static spinal compression leads to an increase in temporal summation of noxious reflex activity, warranting further investigation into the effect of SCI compression on spinal nociception. It is important to note that classical central sensitization was not evaluated in this study (Woolf, 1996; Li et al., 1999; Herrero et al., 2000), which would have been evident by analysing noxious reflex activity after the temporal summation protocol. However, the pronociceptive effect of the T3 spinal conditioning stimulus on prolonged noxious toes-TA reflex activity observed in maLPA1-null mice after SCI following repeated C-fibre stimulation (see Fig. 3B and D) suggests that central sensitization to noxious input is present after compression SCI. The functional switch observed from descending inhibition to facilitation of noxious spinal reflex excitability following thoracic dorsolateral funiculus damage suggests that this mouse model of SCI should be instrumental in revealing both spinal and supraspinal pathophysiological mechanisms related to the development of the spasticity syndrome and local mechanisms of nociception, which are prevalent following SCI (Avila-Martin et al., 2011). However it is not possible in this study to exclude the contribution of intraspinal control systems organized within the low thoracic or upper lumbar spinal level to greater temporal summation of noxious reflex activity after SCI following activation of descending modulatory pathways (Cadden et al., 1983; Cavallari and Pettersson, 1989; Magnuson et al., 1999). Further examination of the balance and integrity of competing descending inhibitory and facilitatory modulatory pain pathways from the brainstem should be assessed as important control mechanisms of spinal reflex excitability following SCI (Gozariu et al., 1997). In this regard, in direct assessment of the role of descending intraspinal modulatory pathways activated either by paraspinal transcutaneous conditioning stimulation in the rat (Avila-Martin et al., 2011) or by non-invasive techniques in man (Albu et al., 2013) may provide important methodological and mechanistic information regarding the state of these modulatory mechanisms following SCI.
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4.5 LPA1 modulation of the immune system after SCI A confounding factor that could account for the functional effects of the compression SCI in the maLPA1-null mice is the possible effect of LPA1 deficiency on the immune component activated after injury. Activated T-cells are known to express LPA1 and this mechanism is able to regulate their proliferation (Huang et al., 2002). Moreover, LPA1 KO mice are resistant to arthritic disease induction which results from a block of immune cell infiltration (Miyabe et al., 2013). Since T-cells play an important role following traumatic SCI, particularly in modulating inflammation, neurodegeneration and repair (Ankeny and Popovich, 2009), the maladaptive pronociceptive response observed in the maLPA1-null mice in this study may also reflect a blunting of repair processes due to disruption of lymphocyte activation following injury. Future studies of the role of LPA receptors in modulation of sensorimotor function following SCI should also assess the role of T-cell activation as a possible contributing factor.
5. Conclusion Maintenance of long-tract descending antinociceptive modulatory pathways is mediated by the LPA1 receptor following thoracic SCI, which plays a role in the control of phasic inhibition of spinal noxious reflex activity and locomotor recovery. Therefore, this study suggests that the LPA1 receptor plays an important role in neuroprotection of descending modulatory pathways following SCI, possibly by maintaining or inducing remyelination of neuronal long-tract systems across the injury site (Weiner et al., 1998; Ye et al., 2002b; Matsushita et al., 2005; Nogaroli et al., 2009; Anliker et al., 2013). Author contributions All authors were responsible for: (1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be published.
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