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Effects of peripheral nerve injury on parvalbumin expression in adult rat dorsal root ganglion neurons

Effects of peripheral nerve injury on parvalbumin expression in adult rat dorsal root ganglion... Background: Parvalbumin (PV ) is a calcium binding protein that identifies a subpopulation of proprioceptive dorsal root ganglion (DRG) neurons. Calcitonin gene-related peptide (CGRP) is also expressed in a high proportion of muscle afferents but its relationship to PV is unclear. Little is known of the phenotypic responses of muscle afferents to nerve injury. Sciatic nerve axotomy or L5 spinal nerve ligation and section (SNL) lesions were used to explore these issues in adult rats using immunocytochemistry. Results: In naive animals, the mean PV expression was 25 % of L4 or L5 dorsal root ganglion (DRG) neurons, and this was unchanged 2 weeks after sciatic nerve axotomy. Colocalization studies with the injury marker activating tran- scription factor 3 (ATF3) showed that approximately 24 % of PV neurons expressed ATF3 after sciatic nerve axotomy suggesting that PV may show a phenotypic switch from injured to uninjured neurons. This possibility was further assessed using the spinal nerve ligation (SNL) injury model where injured and uninjured neurons are located in differ - ent DRGs. Two weeks after L5 SNL there was no change in total PV staining and essentially all L5 PV neurons expressed ATF3. Additionally, there was no increase in PV-ir in the adjacent uninjured L4 DRG cells. Co-labelling of DRG neurons revealed that less than 2 % of PV neurons normally expressed CGRP and no colocalization was seen after injury. Conclusion: These experiments clearly show that axotomy does not produce down regulation of PV protein in the DRG. Moreover, this lack of change is not due to a phenotypic switch in PV immunoreactive (ir) neurons, or de novo expression of PV-ir in uninjured neurons after nerve injury. These results further illustrate differences that occur when muscle afferents are injured as compared to cutaneous afferents. Keywords: Plasticity, Axotomy, Spinal nerve ligation, Dorsal root ganglion, Proprioceptor BDNF, galanin whilst adjacent uninjured neurons can Background increase their content of neurotransmitters such as sub- Peripheral nerve injury disconnects sensory and motor stance P, CGRP, BDNF, galanin and ion channels such axons from their peripheral targets and results in the as TRPRV1 and P2X3 [3–5]. This plasticity is thought production of regeneration associated genes such as α-tubulin, GAP43, CAP23, ATF3, and STAT3 that are to contribute to the generation and maintenance of neu- important in the growth and functional recovery of dam ropathic pain [3, 6, 7]. However, the mechanisms that contribute to chronic pain syndromes are incompletely aged sensory and motor axons [1, 2]. Primary sensory understood. Several different models have been devel - neurons also show considerable plasticity when subjected oped to explore the contributions of primary afferents to to peripheral nerve injury. For example, directly injured neurons can upregulate neurotransmitters such as NPY, chronic pain syndromes [3, 8, 9]. Most models employ injury to the sciatic nerve, a mixed peripheral nerve, or its branches. Interestingly, one study demonstrates the *Correspondence: p.shortland@uws.edu.au importance of muscle afferents to the pathobiology of School of Science and Health, Western Sydney University, Narellen Road, nerve injury pain. When the gastrocnemius (muscle) or Campbelltown, NSW 2560, Australia Full list of author information is available at the end of the article © 2015 Medici and Shortland. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Medici and Shortland BMC Neurosci (2015) 16:93 Page 2 of 10 tibial (mixed) or sural (cutaneous) nerve was sectioned, Therefore, the aim of this study is to test the hypothesis mechanical and thermal hypersensitivity only occurred that peripheral nerve injury induces a phenotypic switch in nerve injuries involving muscle afferents [10]. in subpopulations of PV-ir neurons in the dorsal root The phenotypic responses of muscle afferents to nerve ganglia. injury have been relatively little studied. In the DRG, muscle afferents are generally identified by their size, Results neurotrophic factor dependence, or their expression of Naive animals particular neurotransmitters or proteins [11–18]. Retro- PV-ir was expressed primarily in medium to large-sized grade tracing experiments show that muscle afferents are DRG neurons (Fig.  1a). Quantification of expression in both myelinated and unmyelinated [16]. Muscle nocic- the L4 and L5 ganglia is shown in Table  1. Similar per- eptors are generally small and contain high levels of the centages were expressed in both ganglia and there was neuropeptide CGRP [15, 16] whereas muscle spindle no statistical difference between the ganglia (t   =  0.255, afferents are generally large-sized, myelinated fibres that P > 0.5, 2-tailed test). express carbonic anhydrase, or the calcium binding pro- CGRP-ir was predominantly expressed in small-sized teins calretinin, calbindin, neurocalcin or parvalbumin DRG neurons with some expression in medium- and [19–25]. Some of these markers, however, are not specific large-sized DRGs (Fig.  1b). Quantification of CGRP- to muscle afferents, as they are also found in skin affer - ir expression in the naive L5 ganglia was 48.3  ±  1.9  % ents [26–28]. Whilst parvalbumin is generally regarded (N  =  6). Colocalisation between PV-ir and CGRP- as a reliable marker for proprioceptive afferents [22, 23], ir within the L5 DRG was negligible (1.9  ±  1.9  %) and, genetic studies have revealed that, by itself, it is not a when present, was restricted to the large-sized neurons. selective marker of muscle proprioceptors nor a marker of muscle afferent nociceptors [26, 29]. Sciatic nerve axotomy Relatively few studies have assessed the effects of nerve The expression of PV-ir in the L4 and L5 ganglia 2 weeks injury on parvalbumin expression. Most of the published after sciatic nerve axotomy is detailed in Table 1. The per - literature concerns injuries to branches of the trigemi- centage expression in these ganglia was not significantly nal [30, 31] or sciatic [22, 32, 33] nerves and the gen- different from naïve ganglia (L5 DRG: F   =  1.800, 2,11 eral consensus appears to be that injury has little effect P  >  0.1, L4 DRG: t   =  0.766, P  >  0.5, 2-tailed test). The on parvalbumin expression in the affected DRGs. One PV-ir in axotomised neurons was observed in the possible reason for this is that a phenotypic change in medium to large-sized cells of the ganglia, identical to expression in injured versus uninjured afferents occurs, that seen in naive ganglia. such that parvalbumin expression is decreased in injured It is possible that the unchanged percentage of PV-ir neurons and increased in uninjured neurons. As the sci- after sciatic nerve axotomy reflects a phenotypic switch atic nerve contains axons originating in the L4-6 DRGs, in neurons such that injured neurons lose immunoreac- afferents from these ganglia are only partially affected tivity and uninjured ones start to express PV de novo. To by peripheral nerve injury. By assessing changes only in explore this possibility colocalisation with ATF3 was per- parvalbumin expression with this model it is not pos- formed (Fig. 2). ATF3-ir was expressed in 50.7 % ± 4.9 of sible to tell whether a phenotypic change has occurred. ipsilateral L5 DRG and 43.4 % ± 7.0 of the ipsilateral L4 Phenotypic switching in primary afferents is known to ganglion cells, and these values were was not significantly occur in nerve injury models [3, 5, 34]. This issue can be different from each other (p = 0.21). explored by using the regeneration associated transcrip- If a phenotypic switch occurs, one might expect to see tion factor ATF3 [35]. ATF3 is not expressed in the DRG little or no colocalisation with ATF3 after injury. How- nuclei of uninjured neurons but is rapidly induced in all ever, this was not the case (Fig.  2). Analysis of double injured neurons following peripheral axotomy or spinal labelled sections showed that 23.7 % ± 3.7 of PV-ir cells nerve injury [35]. By quantitatively assessing the number (range 16–30 %) expressed ATF3 in the injured ipsilateral of co-labelled ATF3 and parvalbumin neurons after sci- L4-5 ganglia. By itself, this result is insufficient to deter - atic nerve injury, it is possible to determine the presence mine if a phentotypic switch is occurring. or absence of a phenotypic switch. A second, and more clear-cut, method involves using a spinal nerve ligation Spinal nerve ligation and section injury [36]. This essentially injures all axons To better explore the possibility of a phenotypic switch in the L5 DRG but leaves axons in the adjacent L4 DRG after injury, the experiment was repeated using the L5 largely unaffected [5, 34, 35]. Changes can then be com- spinal nerve injury model, which essentially damages all pared by assessing the numbers of PV cells in the two of the axons of the L5 ganglion neurons whilst leaving adjacent ganglia. most of the axons of the adjacent L4 DRG intact [5, 34]. Medici and Shortland BMC Neurosci (2015) 16:93 Page 3 of 10 Fig. 1 An example of parvalbumin and CGRP staining in naïve L5 DRG. a shows PV-ir (green), b shows CGRP-ir (red) staining and c is the merged image showing essentially no overlap between these populations. Scale bar 100 μm Table 1 Percentage expression of  parvalbumin in  L4 This study found that 25 % of naïve rat L4-5 DRGs were and L5 DRGs in naïve and sciatic axotomy groups PV-ir, consistent with these earlier reports. By itself, PV is not an exclusive marker of muscle proprioceptive affer - L4 DRG L5 DRG ents, as it is also expressed in Pacinian corpuscles, Mer- Naïve (N = 6) 25.7 % ± 1.8 25.1 % ± 1.8 kel cells and lanceolate endings of cutaneous afferents 2 week sciatic nerve axotomy (N = 5) 23.1 % ± 1.9 21.7 % ± 1.5 [26]. Thus, since retrograde tracing from muscles was not used in this study we cannot be sure that all PV-ir cells in this study are muscle proprioceptors. However, only around 1.5  % of L4-5 PV-ir DRG cells arise from skin The L4 and L5 ganglia from both ipsilateral and con - afferents [22] and a recent anatomical study showed that tralateral sides to the L5 SNL were stained (Fig.  3) and 27  % of lumbar dorsal root fibres were positive for α3 quantified for immunoreactivity to PV, ATF3, and CGRP Na+/K+  ATPase, a specific marker of muscle afferents (Table 2). The mean percentage expression of PV-ir in the [13]. The similarity in percentage expression of PV-ir and ipsilateral injured L5 ganglia was not significantly differ - α3 Na+/K+  ATPase implies that PV-ir afferents should ent from naive animals (F  = 0.266, P > 0.5) or the con- 1,8 express this ATPase transporter, although this awaits tralateral side (F  =  2.126, P  >  0.1) or the ipsilateral L4 1,8 confirmation. Together, these suggest that the vast major - DRG (F  = 0.103, P > 0.5). 1,8 ity of PV-ir cells are muscle proprioceptors. ATF3-ir in the contralateral L5 DRG was negligible Whilst PV is almost exclusively expressed in large (Table  2). In the injured L5 ganglia, ATF3-ir was found diameter muscle afferents [22], CGRP is expressed in in almost all cells (Fig.  3), confirming the completeness both large and small diameter muscle afferents [16]. of the lesion. Essentially, nearly all PV-ir neurons coex- The large-sized neurons correspond to the group I-II pressed ATF3 (Table  2). In the ipsilateral L4 ganglia the muscle spindle and Golgi tendon organ (propriocep- average ATF3-ir was low (Table 2) but significantly more tive) afferents, whereas group III-IV afferents are small than the contralateral side (F  = 7.242, P = 0.036), with 1,6 diameter muscle afferents associated with muscle meta - the occasional PV-IR cell showing ATF3 staining (Fig. 3). bolic responses, blood vessel innervation, sensations CGRP staining was essentially absent from the L5 involved during exercise and muscle pain [39]. Colo- DRG neurons in the injured ganglion, although some calisation studies suggest that PV is found in DRGs that axonal staining could be detected (Fig. 4). The character - may also contain calbindin, carbonic anhydrase (CA) and istic punctate staining in the cell cytoplasm that is nor- cytochrome oxidase [11, 20]. This is consistent with the mally present (Fig. 4b) is absent in the ipsilateral L5 DRG proposed role of PV in facilitation of fast, repeated, affer - (Fig.  4a). In the ipsilateral L4 ganglia the average CGRP- ent firing of fast twitch muscles [40, 41]. CGRP is also ir was not significantly different from naïve animals expressed in large-sized DRG cells but the colocalisation (Table  2; p  >  0.5) and colocalisation of CGRP in PV-ir of PV and CGRP was minimal in the present study, con- cells was 0.6 ± 0.3 %. firming earlier studies [23, 42]. Discussion Plasticity of primary afferents after injury PV is often used as a marker of a subpopulation of (mus- DRG cells show considerable plasticity in response to cle) proprioceptive afferents, comprising 10–33 % of cells peripheral nerve injury. Following injury, peptides such in spinal and trigeminal ganglia [11, 19, 22, 23, 33, 37, 38]. Medici and Shortland BMC Neurosci (2015) 16:93 Page 4 of 10 Fig. 2 Parvalbumin (green) and ATF3 (red) staining in the ipsilateral L4 (A, C, E) and L5 (B, D, F) ganglia of rats with sciatic nerve axotomy 2 weeks previously. Arrows indicate double-labelled cells, whilst arrowheads denote cells only expressing ATF3. Both injured and uninjured cells express PV-ir. Scale bar 75 μm as CGRP and substance P are often down regulated in contains cell bodies of cutaneous afferents and the mes - injured DRGs whilst other peptides such as galanin and encephalic nucleus (Vmes), which contains cell bodies of neuropeptide Y are upregulated (reviewed by [4, 6]). This muscle afferents. When the inferior alveolar nerve was de novo expression also occurs in adjacent uninjured sectioned no change in PV numbers occurred in the Vg DRGs [3].Only a few studies have assessed the effects of [31]. When the masseter nerve was sectioned there was axotomy on PV afferents and these have largely been in a significant drop in PV-ir cells in Vmes between 4 and the trigeminal system [31, 43, 44]. The trigeminal sys - 14 days post injury [43]. However, as there was no base- tem is different from the spinal system in that PV-ir is line control values for PV-ir in Vmes, there is no certainty expressed in both the trigeminal ganglion (Vg), which as to whether there was an overall decrease in expression Medici and Shortland BMC Neurosci (2015) 16:93 Page 5 of 10 Fig. 3 Parvalbumin (green) and ATF3 (red) staining in the ipsilateral L4 (A, C, E) and L5 (B, D, F) ganglia 2 weeks after L5 spinal nerve ligation and section. Arrows denote examples of double-labelled cells; arrowheads denote examples of cells that only express ATF3. Almost all PV-ir cells were ATF3-ir in the L5 ganglion (arrows). Very few ATF3 cells were seen in L4 DRG. Scale bar 75 μm Table 2 Eec ff ts of  L5 spinal nerve ligation on  percentage by day 14 or whether there was an increase which nor- expression of  different neurochemical parameters in  the malised by day 14. Alternatively, since PV is expressed in L4-5 DRG neurons 2 weeks after injury periodontal ligament afferents in the Vg [44], the differ - ence between the two studies may reflect a difference in (N = 5) L4 DRG L5 DRG L5 DRG ipsilateral ipsilateral contralateral the response to injury of cutaneous mechanoreceptors versus muscle afferents. Parvalbumin 23.0 % ± 1.2 22.8 % ± 0.9 22.2 % ± 0.8 In spinal DRGs, previous injury studies suggest that CGRP 44.5 % ± 1.7 0 48.3 % ± 2.7 PV numbers either did not change [22] or were tran- ATF3 2.8 % ± 0.9 92.8 % ± 0.9 0.3 % ± 0.1 siently decreased [32]. These studies were at early Medici and Shortland BMC Neurosci (2015) 16:93 Page 6 of 10 Fig. 4 An example of ATF3 and CGRP staining in L5 DRGs 2 weeks after spinal nerve section. a L5i ganglion showing no CGRPr in ATF3 positive cells. CGRP axons were visible in the tissue (arrows) but the characteristic punctate staining in the cell cytoplasm is absent. b Contralateral ganglion showing many CGRP-ir cells (arrows). The curved arrow shows an example of an ATF3+/CGRP− cell. Scale bar = 50 μm time-points when changes in neurochemical phenotype 3  months in the DRG [52]. SBA is expressed in about are only beginning to start [45]. Additionally, these pre- 50 % of small- and medium-sized muscle DRG cells [18] vious studies did not consider the possibility of a phe- and is extensively co-expressed with CGRP but not CA notypic switch between injured and uninjured neuronal [53]. It is possible that PV levels may decline later on, in populations [3, 34]. This study examined changes at a fashion similar to SBA and CA, but further studies are only 1 time point, 2 weeks after injury. Whilst it is pos- required to verify this; however, we see no change in PV sible that changes occurred before then, previous studies expression at 4  weeks after sciatic injury (Shortland and have shown that peak changes in DRG neurotransmit- Medici, unpublished observations). ter staining occur at 2 weeks post injury [4, 46]. Moreo- Does this mean that large-sized, presumed, muscle ver, the percentage expression of ATF3 positive injured proprioceptive afferents are incapable of phenotypic plas - neurons remains unchanged over the first month after ticity? Evidence suggests that at least some medium-large both types of nerve injury [5, 47]. Therefore, analys - diameter muscle afferents upregulate NPY and galanin ing co-labelling at this time point should maximise the and BDNF [10, 54, 55]. Indeed, Zhou et  al. [10] demon- changes of observing any differences as a result of injury. strated that BDNF was increased in axotomised muscle By using the SNL model and co-labelling ATF3 with PV, but not skin afferents and this was critical to produce this study unequivocally showed that injury did not pro- neuropathic pain. Most recently, Fukuoka et al. [56] have duce a phenotypic switch in PV staining from injured to demonstrated an increase in Nav1.7 in axotomised puta- uninjured neurons, and that PV-ir in DRGs is not regu- tive proprioceptors in DRGs and the gracile nucleus fol- lated by nerve injury. This contrasts with small diameter lowing spinal nerve ligation. Taken together, these studies neurons, whose phenotype changes with nerve injury suggest that different neurotransmitters and receptors due to the loss of target derived trophic factors like NGF may be differentially regulated within single cells. CGRP and BDNF [46]. and substance P have also been reported to increase in These results are also consistent with the lack of change large sized DRG neurons [57, 58]. Whilst mRNA lev- of other markers in large-sized DRG neurons after els have confirmed this, protein expression studies have injury: NF200, a marker of large myelinated afferents, is been less convincing. The majority of studies examining unchanged [45, 46] and α3 Na+/K+ ATPase is reported SP or CGRP-IR changes in the axotomised DRG have not to change in spinal nerve injury up to 1  week post used immunofluorescence cytochemistry [5, 45, 59] and injury [17]. Neuronal p75 levels in muscle afferents are failed to confirm the earlier reports based on mRNA or unchanged after injury [48] as is TRPV2 expression [49]; DAB histochemistry, suggesting differential sensitivity TRPV2 has been shown to be colocalised to some muscle based on methodology. In this study, CGRP-ir was almost afferents [50]. CA, which is also localised to muscle pro - completely depleted in the L5 DRG somas after spinal prioceptive afferents, also shows no change in expression nerve injury, and even if a few large cells increase CGRP after sciatic nerve injury [51]. Lastly, the lectin soybean protein expression these results suggest it is not in PV-ir agglutinin (SBA) is unchanged after injury for at least afferents. Medici and Shortland BMC Neurosci (2015) 16:93 Page 7 of 10 Potential roles for parvalbumin after nerve injury or alternate, possibility is that spontaneous discharges PV is a calcium binding protein responsible for control- may affect sensory processing through up-regulation of ling the levels of intracellular calcium ions [60]. Calcium Nav1.7 in axotomised muscle afferents at the level of the is vital for cell function and its concentration must be gracile nucleus [56]. strictly regulated: too little and the cell cannot perform functions such as neurotransmitter release; too much and Conclusions cellular mechanisms of apoptosis are triggered [61]. Sup- These results show that in two different models of port for PV’s ability to increase cell survival comes from peripheral nerve injury, the percentage of PV in DRG animal studies of ALS pathogenesis. PV is only expressed cells remains unchanged compared to naive or contralat- in subsets of motoneurons such as Onuf ’s nucleus and eral DRGs. Furthermore, by using ATF3 as a marker of the oculomotor nuclei [62–65], areas often spared from injured neurons and by using the spinal nerve ligation cell death in ALS patients. When PV was genetically and section model [5, 34], they unequivocally demon- overexpressed in motoneurons in vitro, they were better strate, for the first time, that this is not the result of a phe - able to withstand concentrations of extracellular calcium notypic switch in expression from injured to uninjured associated with excitotoxic cell death and showed an cells, as numbers of PV-ir cells in the intact L4 DRG and increased ability to survive compared to their wild-type the injured L5 DG remain unaltered. Therefore, PV posi - counterparts [66]. tive afferents do not show the same phenotypic plastic - Peripheral nerve injury also causes cell death as a ity that is characteristic of other neuronal subpopulations result of loss of trophic support [67, 68] that is differ - such as nociceptors [4, 46]. These results further high - entially distributed with respect to peripheral target, light the differential responses of muscle as compared to with a preference for loss of cutaneous versus muscle cutaneous afferents after peripheral nerve injury. afferents [48, 69–71]. PV-ir cells are known to be trkC positive [12, 26] and levels of NT3 in peripheral tis- Methods sues are higher at birth then they are in the adult. It is All experimental procedures were carried out in accord- possible that PV-ir cells maintain their trophic support ance with the UK Scientific Procedures Act (1986) and from the CNS where levels are higher [72]. Supporting guidelines set out by the International Association for the this, neonatal nerve crush depletes PV-ir cells from the Study of Pain guidelines for the care and use of animals DRGs [73]. Given that adult axotomy does not deplete [78]. neuronal p75 and that BDNF is upregulated in trkC neu- rons [48, 55], it is probable that PV-ir cells are trophi- Surgery cally supported by BDNF released from BDNF baskets Sixteen adult Wistar rats (250–350  g) were used and that surround trkC cells containing p75 in an autocrine split into 3 groups: naive (n  =  6), sciatic nerve axotomy manner [51]. In contrast, injured small sized, cutaneous (n = 5) and L5 spinal nerve ligation and section (n =  5). DRGs, lose p75 neuronal staining and do not upregulate All surgical procedures were carried out on the left side BDNF after injury [10, 48] and so may be more vulner- under sterile conditions as described in detail previously able to death. [3, 43]. Briefly, the sciatic nerve was exposed at mid-thigh Nerve injury also generates spontaneous and ectopic level, ligated with a 4-0 nylon suture and cut distal to the activity that is associated with axotomised and intact suture. For the spinal nerve injury, the L5 spinal nerve muscle, but not skin, afferents [39, 74, 75] that starts was exposed, ligated with a 4-0 nylon suture and the within hours, lasts many weeks and is predominantly nerve cut distal to it. Following nerve section, the wound associated with A-fibres. As PV-ir is associated with was closed and topical antibiotics applied to the wound fast twitch muscles it is tempting to speculate that it is site to prevent post-operative infection. All animals sur- these fibres that may be spontaneously active in the DRG. vived post-operatively for 2 weeks before perfusion. However, electrophysiological data suggests that the Rats were terminally anaesthetised and perfused with very large-sized DRG cells are not spontaneously active saline followed by 4  % paraformaldehyde (dissolved in [75]. As more than 80  % of axotomised muscle afferents 0.1  M phosphate buffer, pH7.4) fixative. The L4 and L5 express BDNF after injury [10] it is likely that many of DRGs were removed bilaterally and post-fixed for 2  h the PV-ir afferents will express BDNF. Since BDNF is a before being cryoprotected overnight in 30  % sucrose potent neuromodulator of neuronal activity [76, 77], it is dissolved in phosphate buffered saline (PBS) solution. BDNF that is the major contributor to pain states from Ganglia were embedded in OCT (VMR International) muscle afferents [10] whereas PV may be important and frozen prior to sectioning for immunohistochemical for the survival of neurons after injury. An additional, staining. Medici and Shortland BMC Neurosci (2015) 16:93 Page 8 of 10 Abbreviations Immunocytochemistry ir: immunoreactivity; ATF3: activating transcription factor 3; PV: parvalbumin; Longitudinal sections (8–10  µm) were cut on a cryostat CGRP: calcitonin gene related peptide; FITC: fluorescein isothiocyanate; Cy3: and thaw-mounted onto Superfrost plus (VWR Inter- cyanine dye 3; TRITC: tetrarhodamine isothiocyanate; DAPI: 4,6-diamino- 2-phenylindole; DRG: dorsal root ganglion; IgG: immunoglobulin. national) slides. Each adjacent section was separated by 50–100 µm. Sections were blocked in 10 % normal don- Authors’ contributions key serum (Chemicon) for an hour and then incubated PS performed surgery, perfusions and tissue removal. TM performed all histol- ogy, data quantification, statistical analysis, and image capture. Both authors with goat anti-parvalbumin (1:8000, Swant, PVG214 contributed to manuscript writing. Both authors read and approved the final RRID:AB_10000345) for 24 h at room temperature. Slides manuscript. were then washed 3 times in PBS before incubation with Author details fluorescein isothiocyanate (FITC)-conjugated donkey School of Science and Health, Western Sydney University, Narellen Road, anti-goat IgG (Jackson ImmunoResearch, 1:400) for 1 h. Campbelltown, NSW 2560, Australia. Centre for Neuroscience and Trauma, In double labelling studies, sections were first incu - Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Newark Street, London E1 2AT, UK. Present bated in PV for 24  h, washed in PBS and then incu- Address: Queens Hospital, Romford, Essex RM7 0AG, UK. bated with rabbit anti-ATF3 (1:200, Santa Cruz SC-188 RRID:AB_2258513), or rabbit anti-CGRP (1:2000, Acknowledgements Dr Medici was supported by the Worshipful Company of Tallow Chandlers Affinity Research Products Ltd., UK) for 24–48  h at during this study. This work was funded by internal research funds associated room temperature. Following three PBS washes, sec- with the Neuroscience intercalated BSc. Degree at Queen Mary University of tions were then incubated in donkey anti-rabbit Cy3 London. (1:800, Jackson ImmunoResearch, USA) for 2 h at room Competing interests temperature. In some slides, sections were also coun- The authors declare that they have no competing interests. terstained with 100  µg/ml DAPI (4,6-diamino-2-phe- Received: 30 July 2015 Accepted: 7 December 2015 nylindole, Sigma, UK) to reveal neuronal nuclei. All slides were cover-slipped with 8:1 glycerol:PBS solu- tion. Controls for double labelling included reversing the order of the antibodies or omission of the primary or secondary antibodies. The characteristics and valid - References 1. Fagoe ND, Attwell CL, Kouwenhoven D, Verhaagen J, Mason MRJ. 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Delayed loss of small dorsal root science. 2010;166:942–51. ganglion cells after transection of the rat sciatic nerve. J Comp Neurol. 2000;422:172–80. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Neuroscience Springer Journals

Effects of peripheral nerve injury on parvalbumin expression in adult rat dorsal root ganglion neurons

BMC Neuroscience , Volume 16 (1) – Dec 16, 2015

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Springer Journals
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Copyright © 2015 by Medici and Shortland.
Subject
Biomedicine; Neurosciences; Neurobiology; Animal Models
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1471-2202
DOI
10.1186/s12868-015-0232-9
pmid
26674138
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Abstract

Background: Parvalbumin (PV ) is a calcium binding protein that identifies a subpopulation of proprioceptive dorsal root ganglion (DRG) neurons. Calcitonin gene-related peptide (CGRP) is also expressed in a high proportion of muscle afferents but its relationship to PV is unclear. Little is known of the phenotypic responses of muscle afferents to nerve injury. Sciatic nerve axotomy or L5 spinal nerve ligation and section (SNL) lesions were used to explore these issues in adult rats using immunocytochemistry. Results: In naive animals, the mean PV expression was 25 % of L4 or L5 dorsal root ganglion (DRG) neurons, and this was unchanged 2 weeks after sciatic nerve axotomy. Colocalization studies with the injury marker activating tran- scription factor 3 (ATF3) showed that approximately 24 % of PV neurons expressed ATF3 after sciatic nerve axotomy suggesting that PV may show a phenotypic switch from injured to uninjured neurons. This possibility was further assessed using the spinal nerve ligation (SNL) injury model where injured and uninjured neurons are located in differ - ent DRGs. Two weeks after L5 SNL there was no change in total PV staining and essentially all L5 PV neurons expressed ATF3. Additionally, there was no increase in PV-ir in the adjacent uninjured L4 DRG cells. Co-labelling of DRG neurons revealed that less than 2 % of PV neurons normally expressed CGRP and no colocalization was seen after injury. Conclusion: These experiments clearly show that axotomy does not produce down regulation of PV protein in the DRG. Moreover, this lack of change is not due to a phenotypic switch in PV immunoreactive (ir) neurons, or de novo expression of PV-ir in uninjured neurons after nerve injury. These results further illustrate differences that occur when muscle afferents are injured as compared to cutaneous afferents. Keywords: Plasticity, Axotomy, Spinal nerve ligation, Dorsal root ganglion, Proprioceptor BDNF, galanin whilst adjacent uninjured neurons can Background increase their content of neurotransmitters such as sub- Peripheral nerve injury disconnects sensory and motor stance P, CGRP, BDNF, galanin and ion channels such axons from their peripheral targets and results in the as TRPRV1 and P2X3 [3–5]. This plasticity is thought production of regeneration associated genes such as α-tubulin, GAP43, CAP23, ATF3, and STAT3 that are to contribute to the generation and maintenance of neu- important in the growth and functional recovery of dam ropathic pain [3, 6, 7]. However, the mechanisms that contribute to chronic pain syndromes are incompletely aged sensory and motor axons [1, 2]. Primary sensory understood. Several different models have been devel - neurons also show considerable plasticity when subjected oped to explore the contributions of primary afferents to to peripheral nerve injury. For example, directly injured neurons can upregulate neurotransmitters such as NPY, chronic pain syndromes [3, 8, 9]. Most models employ injury to the sciatic nerve, a mixed peripheral nerve, or its branches. Interestingly, one study demonstrates the *Correspondence: p.shortland@uws.edu.au importance of muscle afferents to the pathobiology of School of Science and Health, Western Sydney University, Narellen Road, nerve injury pain. When the gastrocnemius (muscle) or Campbelltown, NSW 2560, Australia Full list of author information is available at the end of the article © 2015 Medici and Shortland. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons. org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Medici and Shortland BMC Neurosci (2015) 16:93 Page 2 of 10 tibial (mixed) or sural (cutaneous) nerve was sectioned, Therefore, the aim of this study is to test the hypothesis mechanical and thermal hypersensitivity only occurred that peripheral nerve injury induces a phenotypic switch in nerve injuries involving muscle afferents [10]. in subpopulations of PV-ir neurons in the dorsal root The phenotypic responses of muscle afferents to nerve ganglia. injury have been relatively little studied. In the DRG, muscle afferents are generally identified by their size, Results neurotrophic factor dependence, or their expression of Naive animals particular neurotransmitters or proteins [11–18]. Retro- PV-ir was expressed primarily in medium to large-sized grade tracing experiments show that muscle afferents are DRG neurons (Fig.  1a). Quantification of expression in both myelinated and unmyelinated [16]. Muscle nocic- the L4 and L5 ganglia is shown in Table  1. Similar per- eptors are generally small and contain high levels of the centages were expressed in both ganglia and there was neuropeptide CGRP [15, 16] whereas muscle spindle no statistical difference between the ganglia (t   =  0.255, afferents are generally large-sized, myelinated fibres that P > 0.5, 2-tailed test). express carbonic anhydrase, or the calcium binding pro- CGRP-ir was predominantly expressed in small-sized teins calretinin, calbindin, neurocalcin or parvalbumin DRG neurons with some expression in medium- and [19–25]. Some of these markers, however, are not specific large-sized DRGs (Fig.  1b). Quantification of CGRP- to muscle afferents, as they are also found in skin affer - ir expression in the naive L5 ganglia was 48.3  ±  1.9  % ents [26–28]. Whilst parvalbumin is generally regarded (N  =  6). Colocalisation between PV-ir and CGRP- as a reliable marker for proprioceptive afferents [22, 23], ir within the L5 DRG was negligible (1.9  ±  1.9  %) and, genetic studies have revealed that, by itself, it is not a when present, was restricted to the large-sized neurons. selective marker of muscle proprioceptors nor a marker of muscle afferent nociceptors [26, 29]. Sciatic nerve axotomy Relatively few studies have assessed the effects of nerve The expression of PV-ir in the L4 and L5 ganglia 2 weeks injury on parvalbumin expression. Most of the published after sciatic nerve axotomy is detailed in Table 1. The per - literature concerns injuries to branches of the trigemi- centage expression in these ganglia was not significantly nal [30, 31] or sciatic [22, 32, 33] nerves and the gen- different from naïve ganglia (L5 DRG: F   =  1.800, 2,11 eral consensus appears to be that injury has little effect P  >  0.1, L4 DRG: t   =  0.766, P  >  0.5, 2-tailed test). The on parvalbumin expression in the affected DRGs. One PV-ir in axotomised neurons was observed in the possible reason for this is that a phenotypic change in medium to large-sized cells of the ganglia, identical to expression in injured versus uninjured afferents occurs, that seen in naive ganglia. such that parvalbumin expression is decreased in injured It is possible that the unchanged percentage of PV-ir neurons and increased in uninjured neurons. As the sci- after sciatic nerve axotomy reflects a phenotypic switch atic nerve contains axons originating in the L4-6 DRGs, in neurons such that injured neurons lose immunoreac- afferents from these ganglia are only partially affected tivity and uninjured ones start to express PV de novo. To by peripheral nerve injury. By assessing changes only in explore this possibility colocalisation with ATF3 was per- parvalbumin expression with this model it is not pos- formed (Fig. 2). ATF3-ir was expressed in 50.7 % ± 4.9 of sible to tell whether a phenotypic change has occurred. ipsilateral L5 DRG and 43.4 % ± 7.0 of the ipsilateral L4 Phenotypic switching in primary afferents is known to ganglion cells, and these values were was not significantly occur in nerve injury models [3, 5, 34]. This issue can be different from each other (p = 0.21). explored by using the regeneration associated transcrip- If a phenotypic switch occurs, one might expect to see tion factor ATF3 [35]. ATF3 is not expressed in the DRG little or no colocalisation with ATF3 after injury. How- nuclei of uninjured neurons but is rapidly induced in all ever, this was not the case (Fig.  2). Analysis of double injured neurons following peripheral axotomy or spinal labelled sections showed that 23.7 % ± 3.7 of PV-ir cells nerve injury [35]. By quantitatively assessing the number (range 16–30 %) expressed ATF3 in the injured ipsilateral of co-labelled ATF3 and parvalbumin neurons after sci- L4-5 ganglia. By itself, this result is insufficient to deter - atic nerve injury, it is possible to determine the presence mine if a phentotypic switch is occurring. or absence of a phenotypic switch. A second, and more clear-cut, method involves using a spinal nerve ligation Spinal nerve ligation and section injury [36]. This essentially injures all axons To better explore the possibility of a phenotypic switch in the L5 DRG but leaves axons in the adjacent L4 DRG after injury, the experiment was repeated using the L5 largely unaffected [5, 34, 35]. Changes can then be com- spinal nerve injury model, which essentially damages all pared by assessing the numbers of PV cells in the two of the axons of the L5 ganglion neurons whilst leaving adjacent ganglia. most of the axons of the adjacent L4 DRG intact [5, 34]. Medici and Shortland BMC Neurosci (2015) 16:93 Page 3 of 10 Fig. 1 An example of parvalbumin and CGRP staining in naïve L5 DRG. a shows PV-ir (green), b shows CGRP-ir (red) staining and c is the merged image showing essentially no overlap between these populations. Scale bar 100 μm Table 1 Percentage expression of  parvalbumin in  L4 This study found that 25 % of naïve rat L4-5 DRGs were and L5 DRGs in naïve and sciatic axotomy groups PV-ir, consistent with these earlier reports. By itself, PV is not an exclusive marker of muscle proprioceptive affer - L4 DRG L5 DRG ents, as it is also expressed in Pacinian corpuscles, Mer- Naïve (N = 6) 25.7 % ± 1.8 25.1 % ± 1.8 kel cells and lanceolate endings of cutaneous afferents 2 week sciatic nerve axotomy (N = 5) 23.1 % ± 1.9 21.7 % ± 1.5 [26]. Thus, since retrograde tracing from muscles was not used in this study we cannot be sure that all PV-ir cells in this study are muscle proprioceptors. However, only around 1.5  % of L4-5 PV-ir DRG cells arise from skin The L4 and L5 ganglia from both ipsilateral and con - afferents [22] and a recent anatomical study showed that tralateral sides to the L5 SNL were stained (Fig.  3) and 27  % of lumbar dorsal root fibres were positive for α3 quantified for immunoreactivity to PV, ATF3, and CGRP Na+/K+  ATPase, a specific marker of muscle afferents (Table 2). The mean percentage expression of PV-ir in the [13]. The similarity in percentage expression of PV-ir and ipsilateral injured L5 ganglia was not significantly differ - α3 Na+/K+  ATPase implies that PV-ir afferents should ent from naive animals (F  = 0.266, P > 0.5) or the con- 1,8 express this ATPase transporter, although this awaits tralateral side (F  =  2.126, P  >  0.1) or the ipsilateral L4 1,8 confirmation. Together, these suggest that the vast major - DRG (F  = 0.103, P > 0.5). 1,8 ity of PV-ir cells are muscle proprioceptors. ATF3-ir in the contralateral L5 DRG was negligible Whilst PV is almost exclusively expressed in large (Table  2). In the injured L5 ganglia, ATF3-ir was found diameter muscle afferents [22], CGRP is expressed in in almost all cells (Fig.  3), confirming the completeness both large and small diameter muscle afferents [16]. of the lesion. Essentially, nearly all PV-ir neurons coex- The large-sized neurons correspond to the group I-II pressed ATF3 (Table  2). In the ipsilateral L4 ganglia the muscle spindle and Golgi tendon organ (propriocep- average ATF3-ir was low (Table 2) but significantly more tive) afferents, whereas group III-IV afferents are small than the contralateral side (F  = 7.242, P = 0.036), with 1,6 diameter muscle afferents associated with muscle meta - the occasional PV-IR cell showing ATF3 staining (Fig. 3). bolic responses, blood vessel innervation, sensations CGRP staining was essentially absent from the L5 involved during exercise and muscle pain [39]. Colo- DRG neurons in the injured ganglion, although some calisation studies suggest that PV is found in DRGs that axonal staining could be detected (Fig. 4). The character - may also contain calbindin, carbonic anhydrase (CA) and istic punctate staining in the cell cytoplasm that is nor- cytochrome oxidase [11, 20]. This is consistent with the mally present (Fig. 4b) is absent in the ipsilateral L5 DRG proposed role of PV in facilitation of fast, repeated, affer - (Fig.  4a). In the ipsilateral L4 ganglia the average CGRP- ent firing of fast twitch muscles [40, 41]. CGRP is also ir was not significantly different from naïve animals expressed in large-sized DRG cells but the colocalisation (Table  2; p  >  0.5) and colocalisation of CGRP in PV-ir of PV and CGRP was minimal in the present study, con- cells was 0.6 ± 0.3 %. firming earlier studies [23, 42]. Discussion Plasticity of primary afferents after injury PV is often used as a marker of a subpopulation of (mus- DRG cells show considerable plasticity in response to cle) proprioceptive afferents, comprising 10–33 % of cells peripheral nerve injury. Following injury, peptides such in spinal and trigeminal ganglia [11, 19, 22, 23, 33, 37, 38]. Medici and Shortland BMC Neurosci (2015) 16:93 Page 4 of 10 Fig. 2 Parvalbumin (green) and ATF3 (red) staining in the ipsilateral L4 (A, C, E) and L5 (B, D, F) ganglia of rats with sciatic nerve axotomy 2 weeks previously. Arrows indicate double-labelled cells, whilst arrowheads denote cells only expressing ATF3. Both injured and uninjured cells express PV-ir. Scale bar 75 μm as CGRP and substance P are often down regulated in contains cell bodies of cutaneous afferents and the mes - injured DRGs whilst other peptides such as galanin and encephalic nucleus (Vmes), which contains cell bodies of neuropeptide Y are upregulated (reviewed by [4, 6]). This muscle afferents. When the inferior alveolar nerve was de novo expression also occurs in adjacent uninjured sectioned no change in PV numbers occurred in the Vg DRGs [3].Only a few studies have assessed the effects of [31]. When the masseter nerve was sectioned there was axotomy on PV afferents and these have largely been in a significant drop in PV-ir cells in Vmes between 4 and the trigeminal system [31, 43, 44]. The trigeminal sys - 14 days post injury [43]. However, as there was no base- tem is different from the spinal system in that PV-ir is line control values for PV-ir in Vmes, there is no certainty expressed in both the trigeminal ganglion (Vg), which as to whether there was an overall decrease in expression Medici and Shortland BMC Neurosci (2015) 16:93 Page 5 of 10 Fig. 3 Parvalbumin (green) and ATF3 (red) staining in the ipsilateral L4 (A, C, E) and L5 (B, D, F) ganglia 2 weeks after L5 spinal nerve ligation and section. Arrows denote examples of double-labelled cells; arrowheads denote examples of cells that only express ATF3. Almost all PV-ir cells were ATF3-ir in the L5 ganglion (arrows). Very few ATF3 cells were seen in L4 DRG. Scale bar 75 μm Table 2 Eec ff ts of  L5 spinal nerve ligation on  percentage by day 14 or whether there was an increase which nor- expression of  different neurochemical parameters in  the malised by day 14. Alternatively, since PV is expressed in L4-5 DRG neurons 2 weeks after injury periodontal ligament afferents in the Vg [44], the differ - ence between the two studies may reflect a difference in (N = 5) L4 DRG L5 DRG L5 DRG ipsilateral ipsilateral contralateral the response to injury of cutaneous mechanoreceptors versus muscle afferents. Parvalbumin 23.0 % ± 1.2 22.8 % ± 0.9 22.2 % ± 0.8 In spinal DRGs, previous injury studies suggest that CGRP 44.5 % ± 1.7 0 48.3 % ± 2.7 PV numbers either did not change [22] or were tran- ATF3 2.8 % ± 0.9 92.8 % ± 0.9 0.3 % ± 0.1 siently decreased [32]. These studies were at early Medici and Shortland BMC Neurosci (2015) 16:93 Page 6 of 10 Fig. 4 An example of ATF3 and CGRP staining in L5 DRGs 2 weeks after spinal nerve section. a L5i ganglion showing no CGRPr in ATF3 positive cells. CGRP axons were visible in the tissue (arrows) but the characteristic punctate staining in the cell cytoplasm is absent. b Contralateral ganglion showing many CGRP-ir cells (arrows). The curved arrow shows an example of an ATF3+/CGRP− cell. Scale bar = 50 μm time-points when changes in neurochemical phenotype 3  months in the DRG [52]. SBA is expressed in about are only beginning to start [45]. Additionally, these pre- 50 % of small- and medium-sized muscle DRG cells [18] vious studies did not consider the possibility of a phe- and is extensively co-expressed with CGRP but not CA notypic switch between injured and uninjured neuronal [53]. It is possible that PV levels may decline later on, in populations [3, 34]. This study examined changes at a fashion similar to SBA and CA, but further studies are only 1 time point, 2 weeks after injury. Whilst it is pos- required to verify this; however, we see no change in PV sible that changes occurred before then, previous studies expression at 4  weeks after sciatic injury (Shortland and have shown that peak changes in DRG neurotransmit- Medici, unpublished observations). ter staining occur at 2 weeks post injury [4, 46]. Moreo- Does this mean that large-sized, presumed, muscle ver, the percentage expression of ATF3 positive injured proprioceptive afferents are incapable of phenotypic plas - neurons remains unchanged over the first month after ticity? Evidence suggests that at least some medium-large both types of nerve injury [5, 47]. Therefore, analys - diameter muscle afferents upregulate NPY and galanin ing co-labelling at this time point should maximise the and BDNF [10, 54, 55]. Indeed, Zhou et  al. [10] demon- changes of observing any differences as a result of injury. strated that BDNF was increased in axotomised muscle By using the SNL model and co-labelling ATF3 with PV, but not skin afferents and this was critical to produce this study unequivocally showed that injury did not pro- neuropathic pain. Most recently, Fukuoka et al. [56] have duce a phenotypic switch in PV staining from injured to demonstrated an increase in Nav1.7 in axotomised puta- uninjured neurons, and that PV-ir in DRGs is not regu- tive proprioceptors in DRGs and the gracile nucleus fol- lated by nerve injury. This contrasts with small diameter lowing spinal nerve ligation. Taken together, these studies neurons, whose phenotype changes with nerve injury suggest that different neurotransmitters and receptors due to the loss of target derived trophic factors like NGF may be differentially regulated within single cells. CGRP and BDNF [46]. and substance P have also been reported to increase in These results are also consistent with the lack of change large sized DRG neurons [57, 58]. Whilst mRNA lev- of other markers in large-sized DRG neurons after els have confirmed this, protein expression studies have injury: NF200, a marker of large myelinated afferents, is been less convincing. The majority of studies examining unchanged [45, 46] and α3 Na+/K+ ATPase is reported SP or CGRP-IR changes in the axotomised DRG have not to change in spinal nerve injury up to 1  week post used immunofluorescence cytochemistry [5, 45, 59] and injury [17]. Neuronal p75 levels in muscle afferents are failed to confirm the earlier reports based on mRNA or unchanged after injury [48] as is TRPV2 expression [49]; DAB histochemistry, suggesting differential sensitivity TRPV2 has been shown to be colocalised to some muscle based on methodology. In this study, CGRP-ir was almost afferents [50]. CA, which is also localised to muscle pro - completely depleted in the L5 DRG somas after spinal prioceptive afferents, also shows no change in expression nerve injury, and even if a few large cells increase CGRP after sciatic nerve injury [51]. Lastly, the lectin soybean protein expression these results suggest it is not in PV-ir agglutinin (SBA) is unchanged after injury for at least afferents. Medici and Shortland BMC Neurosci (2015) 16:93 Page 7 of 10 Potential roles for parvalbumin after nerve injury or alternate, possibility is that spontaneous discharges PV is a calcium binding protein responsible for control- may affect sensory processing through up-regulation of ling the levels of intracellular calcium ions [60]. Calcium Nav1.7 in axotomised muscle afferents at the level of the is vital for cell function and its concentration must be gracile nucleus [56]. strictly regulated: too little and the cell cannot perform functions such as neurotransmitter release; too much and Conclusions cellular mechanisms of apoptosis are triggered [61]. Sup- These results show that in two different models of port for PV’s ability to increase cell survival comes from peripheral nerve injury, the percentage of PV in DRG animal studies of ALS pathogenesis. PV is only expressed cells remains unchanged compared to naive or contralat- in subsets of motoneurons such as Onuf ’s nucleus and eral DRGs. Furthermore, by using ATF3 as a marker of the oculomotor nuclei [62–65], areas often spared from injured neurons and by using the spinal nerve ligation cell death in ALS patients. When PV was genetically and section model [5, 34], they unequivocally demon- overexpressed in motoneurons in vitro, they were better strate, for the first time, that this is not the result of a phe - able to withstand concentrations of extracellular calcium notypic switch in expression from injured to uninjured associated with excitotoxic cell death and showed an cells, as numbers of PV-ir cells in the intact L4 DRG and increased ability to survive compared to their wild-type the injured L5 DG remain unaltered. Therefore, PV posi - counterparts [66]. tive afferents do not show the same phenotypic plastic - Peripheral nerve injury also causes cell death as a ity that is characteristic of other neuronal subpopulations result of loss of trophic support [67, 68] that is differ - such as nociceptors [4, 46]. These results further high - entially distributed with respect to peripheral target, light the differential responses of muscle as compared to with a preference for loss of cutaneous versus muscle cutaneous afferents after peripheral nerve injury. afferents [48, 69–71]. PV-ir cells are known to be trkC positive [12, 26] and levels of NT3 in peripheral tis- Methods sues are higher at birth then they are in the adult. It is All experimental procedures were carried out in accord- possible that PV-ir cells maintain their trophic support ance with the UK Scientific Procedures Act (1986) and from the CNS where levels are higher [72]. Supporting guidelines set out by the International Association for the this, neonatal nerve crush depletes PV-ir cells from the Study of Pain guidelines for the care and use of animals DRGs [73]. Given that adult axotomy does not deplete [78]. neuronal p75 and that BDNF is upregulated in trkC neu- rons [48, 55], it is probable that PV-ir cells are trophi- Surgery cally supported by BDNF released from BDNF baskets Sixteen adult Wistar rats (250–350  g) were used and that surround trkC cells containing p75 in an autocrine split into 3 groups: naive (n  =  6), sciatic nerve axotomy manner [51]. In contrast, injured small sized, cutaneous (n = 5) and L5 spinal nerve ligation and section (n =  5). DRGs, lose p75 neuronal staining and do not upregulate All surgical procedures were carried out on the left side BDNF after injury [10, 48] and so may be more vulner- under sterile conditions as described in detail previously able to death. [3, 43]. Briefly, the sciatic nerve was exposed at mid-thigh Nerve injury also generates spontaneous and ectopic level, ligated with a 4-0 nylon suture and cut distal to the activity that is associated with axotomised and intact suture. For the spinal nerve injury, the L5 spinal nerve muscle, but not skin, afferents [39, 74, 75] that starts was exposed, ligated with a 4-0 nylon suture and the within hours, lasts many weeks and is predominantly nerve cut distal to it. Following nerve section, the wound associated with A-fibres. As PV-ir is associated with was closed and topical antibiotics applied to the wound fast twitch muscles it is tempting to speculate that it is site to prevent post-operative infection. All animals sur- these fibres that may be spontaneously active in the DRG. vived post-operatively for 2 weeks before perfusion. However, electrophysiological data suggests that the Rats were terminally anaesthetised and perfused with very large-sized DRG cells are not spontaneously active saline followed by 4  % paraformaldehyde (dissolved in [75]. As more than 80  % of axotomised muscle afferents 0.1  M phosphate buffer, pH7.4) fixative. The L4 and L5 express BDNF after injury [10] it is likely that many of DRGs were removed bilaterally and post-fixed for 2  h the PV-ir afferents will express BDNF. Since BDNF is a before being cryoprotected overnight in 30  % sucrose potent neuromodulator of neuronal activity [76, 77], it is dissolved in phosphate buffered saline (PBS) solution. BDNF that is the major contributor to pain states from Ganglia were embedded in OCT (VMR International) muscle afferents [10] whereas PV may be important and frozen prior to sectioning for immunohistochemical for the survival of neurons after injury. An additional, staining. Medici and Shortland BMC Neurosci (2015) 16:93 Page 8 of 10 Abbreviations Immunocytochemistry ir: immunoreactivity; ATF3: activating transcription factor 3; PV: parvalbumin; Longitudinal sections (8–10  µm) were cut on a cryostat CGRP: calcitonin gene related peptide; FITC: fluorescein isothiocyanate; Cy3: and thaw-mounted onto Superfrost plus (VWR Inter- cyanine dye 3; TRITC: tetrarhodamine isothiocyanate; DAPI: 4,6-diamino- 2-phenylindole; DRG: dorsal root ganglion; IgG: immunoglobulin. national) slides. Each adjacent section was separated by 50–100 µm. Sections were blocked in 10 % normal don- Authors’ contributions key serum (Chemicon) for an hour and then incubated PS performed surgery, perfusions and tissue removal. TM performed all histol- ogy, data quantification, statistical analysis, and image capture. Both authors with goat anti-parvalbumin (1:8000, Swant, PVG214 contributed to manuscript writing. Both authors read and approved the final RRID:AB_10000345) for 24 h at room temperature. Slides manuscript. were then washed 3 times in PBS before incubation with Author details fluorescein isothiocyanate (FITC)-conjugated donkey School of Science and Health, Western Sydney University, Narellen Road, anti-goat IgG (Jackson ImmunoResearch, 1:400) for 1 h. Campbelltown, NSW 2560, Australia. Centre for Neuroscience and Trauma, In double labelling studies, sections were first incu - Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Newark Street, London E1 2AT, UK. Present bated in PV for 24  h, washed in PBS and then incu- Address: Queens Hospital, Romford, Essex RM7 0AG, UK. bated with rabbit anti-ATF3 (1:200, Santa Cruz SC-188 RRID:AB_2258513), or rabbit anti-CGRP (1:2000, Acknowledgements Dr Medici was supported by the Worshipful Company of Tallow Chandlers Affinity Research Products Ltd., UK) for 24–48  h at during this study. This work was funded by internal research funds associated room temperature. Following three PBS washes, sec- with the Neuroscience intercalated BSc. Degree at Queen Mary University of tions were then incubated in donkey anti-rabbit Cy3 London. (1:800, Jackson ImmunoResearch, USA) for 2 h at room Competing interests temperature. In some slides, sections were also coun- The authors declare that they have no competing interests. terstained with 100  µg/ml DAPI (4,6-diamino-2-phe- Received: 30 July 2015 Accepted: 7 December 2015 nylindole, Sigma, UK) to reveal neuronal nuclei. All slides were cover-slipped with 8:1 glycerol:PBS solu- tion. Controls for double labelling included reversing the order of the antibodies or omission of the primary or secondary antibodies. The characteristics and valid - References 1. Fagoe ND, Attwell CL, Kouwenhoven D, Verhaagen J, Mason MRJ. 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BMC NeuroscienceSpringer Journals

Published: Dec 16, 2015

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