- Email: [email protected]
Bone Trauma Causes Massive but Reversible Changes in Spinal Circuitry
Accepted Manuscript Spinal Reorganization after Bone Fracture in Mice Dr. Silke Hirsch, Alaa Ibrahim, Laura Krämer, Fabiola Escolano-Lozano, Dr. Tanja Schlereth, Dr. Frank Birklein, Prof. PII:
S1526-5900(16)30369-8
DOI:
10.1016/j.jpain.2016.12.010
Reference:
YJPAI 3349
To appear in:
Journal of Pain
Received Date: 8 November 2016 Revised Date:
6 December 2016
Accepted Date: 21 December 2016
Please cite this article as: Hirsch S, Ibrahim A, Krämer L, Escolano-Lozano F, Schlereth T, Birklein F, Spinal Reorganization after Bone Fracture in Mice, Journal of Pain (2017), doi: 10.1016/ j.jpain.2016.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Hirsch_manuscript_JP2016
Article type:
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Original article in the section “Neurology” Title:
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Spinal Reorganization after Bone Fracture in Mice
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Running Head:
Bone trauma causes massive but reversible changes in spinal circuitry. Authors: Dr. Silke Hirsch (corresponding author)
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Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1
Germany
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55131 Mainz
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Tel: +49 170 4728743
Email: [email protected]
Alaa Ibrahim
Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1 55131 Mainz Germany 1
ACCEPTED MANUSCRIPT Laura Krämer Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1 55131 Mainz
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Germany
Fabiola Escolano-Lozano
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Klinik und Poliklinik für Neurologie Unimedizin Mainz
55131 Mainz Germany
Dr. Tanja Schlereth
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Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1
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55131 Mainz Germany
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Langenbeckstr. 1
Prof. Dr. Frank Birklein
Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1 55131 Mainz Germany
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ACCEPTED MANUSCRIPT Disclosures: Research funding for this project was provided by the Hopp Foundation, Project 23016006; the DFG, Bi 579/8-1; the EU, FP7 ncRNAPA, project number 602133; the workers compensation insurance BGW
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Mainz (all to FB) and grant no. 97388102 IFF (for SH). There is no conflict of interests for any of the authors. All authors discussed the results and commented on the manuscript.
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Highlights:
Receptive fields are enlarged in the spinal cord after bone trauma in mice.
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The representation area of the injured body part is extended in the murine spinal cord.
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The spinal neuronal changes develop and subside together with the behavioral hypersensitivity.
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Abstract
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Bone fracture with subsequent immobilization of the injured limb can cause Complex Regional Pain
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Syndrome (CRPS) in humans. Mechanisms of CRPS are still not completely understood but bone fracture with casting in mice leads to a similar posttraumatic inflammation as seen in humans and might
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therefore be an analogue to human CRPS. Here we report behavioral and spinal electrophysiological changes in mice that developed swelling of the paw, warming of the skin and pain in the injured limb after bone fracture. The receptive field sizes of spinal neurons representing areas of the hind paws increased after trauma and recovered over time – as did the behavioral signs of inflammation and pain. Interestingly, both sides – the ipsi- and the contralateral limb – showed changes in mechanical sensitivity and neuronal network organization after the trauma. The characteristics of evoked neuronal responses recorded in the dorsal horn of the mice were similar between uninjured controls and fractured animals. However, we saw a caudal extension of the represented area of the hind paw in the spinal cord 3
ACCEPTED MANUSCRIPT at the injured side and an occurrence of large receptive fields of wide dynamic range neurons. The findings in mice compare to human symptoms in CRPS with ipsi- but also contralateral allodynia and pain. In all mice tested, all signs subsided twelve weeks after trauma. Our data suggest a significant
of neuropathies. This process seems to be reversible in the rodent.
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Perspective
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reorganization of spinal circuitry after limb trauma, in a degree more comprehensive than most models
The discovery of enlarged spinal neuronal receptive fields and caudal extension of the representation
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area of the injured body part which subsides several weeks after a bone trauma in mice might give hope to patients of Complex Regional Pain Syndrome if – in the future – we are able to translate the rodent
CRPS
Mouse
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Spinal cord
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Keywords
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recovery mechanisms to posttraumatic humans.
Receptive field
Hypersensitivity
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ACCEPTED MANUSCRIPT Introduction Bone fracture with subsequent immobilization of the injured limb can rarely cause a debilitating, painful syndrome called Complex Regional Pain Syndrome (CRPS) in human patients 40. CRPS is
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characterized by symptoms which go beyond a nerve innervation territory and spread distally. These symptoms are pain and hyperalgesia, signs of inflammation such as skin temperature changes and edema, motor disturbances and trophic changes. The full clinical picture of CRPS seems unique to humans, mainly because of a strong central pathophysiology component which is characterized by brain
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reorganization, body perception disturbances and learned maladaptive behavior. However, mammals
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like mice or rats develop a painful inflammatory phenotype as posttraumatic reaction after a bone fracture which is similar to humans 7. Thus, the tibia fracture mouse model 18,23 is employed here to study posttraumatic changes in behavior and spinal electrophysiology. This model shares posttraumatic inflammatory signs and neuropathic symptoms such as hypersensitivity to mild non-noxious stimuli 45,46 with human CRPS 40. These signs resemble the early inflammatory stage of CRPS and accordingly an
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increase of pro-inflammatory markers such as TNFα, NGF and Il-6 44,57 was found in rat and mouse skin, which has been found in skin biopsies from human CRPS patients as well 5. In contrast to human CRPS, symptoms occur almost regularly and disappear after a few weeks perhaps because of the
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lacking maladaptive learning of rodents.
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The pathophysiology leading to the phenotype in the tibia fracture mouse model is characterized by a massive immune response causing hyperalgesia 23 which is probably mediated by sensitization of primary afferent nociceptors like in other pain models 31 or second order afferent neurons in the spinal cord 45. The neuronal mechanisms underlying the hyperalgesia behavior in fractured mice are not yet specified. Receptive field reorganization is a characteristic of neuronal plasticity after peripheral nerve injury 16,17 and enlargement of receptive field sizes in response to low intensity mechanical stimuli has been suggested as an explanation for the development of allodynia 47. Enlarged receptive fields of spinal 5
ACCEPTED MANUSCRIPT neurons have been described in the chronic constriction injury model 14, after partial sciatic nerve ligation 51 and spinal nerve ligation 47. We therefore hypothesized that one neuronal signature of pain and hyperalgesia behavior of mice with
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distal tibia fracture and cast immobilization of the leg for three weeks may be the enlargement of receptive fields of those spinal neurons that receive afferent input from the fractured limb.
Comprehensive reorganization processes in the dorsal horn have been reported in a rat model of
cancer-induced bone pain 55. With our electrophysiological study we aim to estimate the extent of this
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reorganization and whether this spinal reorganization relates to hyperalgesia behavior.
Material and Methods Mice
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Experiments were conducted with 150 male mice, C57/bl6j. Animals were housed in group cages, food and water ad libitum, twelve hour dark/light cycle, in the institutional animal facilities. All procedures followed the guidelines for animal research of the German Animal Welfare Act 2006 and adhered to the
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guidelines of the Committee for Research and Ethical Issues of IASP 1983. They were approved by the German federal government and supervised by the institutional veterinarian. Two of the animals died
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before the end of experiments; the remaining 148 animals were included in the analysis. Electrophysiological experiments were conducted as the final experiment in the same behaviorally examined animals in order to reduce the number of animals needed. Group sizes were minimized according to precedent power analyses and suffering of animals was reduced to the minimum possible. Experimenters were blinded to the group of investigation. We adhered in sex and age to previous work on the tibia fracture model to make our study comparable to the literature – disregarding the fact that mechanisms might differ between males and females10.
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ACCEPTED MANUSCRIPT Bone Fracture At the age of 6 weeks, mice received a closed tibia fracture of the right leg with subsequent casting for 3 weeks. Surgery was done under isoflurane anaesthesia (2%) combined with buprenorphine analgesia
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during, 12 and 24 hours after surgery (0.05 mg/kg, s.c. each) and antibiosis (enrofloxacin, 5 mg/kg s.c.). To achieve a closed fracture of the shank without rupturing the skin, the leg was tightly held between two fingers; a swab was placed around the lower leg and held in place with an open needle holder. The needle holder was then bent so that the tibia would break once at the spot where the inner edge of the
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needle holder pressed against the swab. The pressure was released immediately after the audible click
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of the bone breaking was heard; the leg was returned to its straight position. In this physiological position the cast was placed around the lower leg, the trunk and the palm of the foot to support the ankle in a 90° position. After 3 weeks, casts were removed under isoflurane anaesthesia.
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Mechanical allodynia, weight bearing, edema and skin temperature measurement Mechanical allodynia was measured with von Frey hairs (Aesthesio, Ugo Basile) using an up-down
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paradigm 12 as previously used for sensitivity testing of bone fractured rodents 20,21. Weight bearing was measured with an incapacitance meter (IITC Life Science) following a protocol also established for
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fractured rodents 21,44. In brief, each hind limb was rested on a separate scale plate measuring the weight put on this leg for 5 s. With an interval of 10 s this measurement was repeated six times. Values were averaged. The incapacitance device has been validated by the group of W. Kingery, Palo Alto, in the mouse fracture model 23,33,48,49. Thickness of the hind paw was measured with a Laser sensor (4381 PreciCura, Limab, Sweden). Skin temperature of the hind paw was measured with a small wire thermometer (OM-CP, Omega, USA) which was placed onto the skin in between the toes 20,21.
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ACCEPTED MANUSCRIPT Electrophysiology A laminectomy was performed under isoflurane anaesthesia (2% during surgery, 1% during recording) to enable extracellular recordings of the activity of single wide dynamic range (WDR) neurons. The
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spinal cord under the vertebrae Th12-L1 was exposed to access the spinal segments innervating the hind paw. Tungsten electrodes (2 MOhm) were inserted into the deep (100-400 µm from dorsal surface) dorsal horn to record identified WDR neurons sorted by spike amplitude and characterized by their responsiveness to mild (6 g), medium (26 g) and strong (80 g) von Frey stimuli. We did not observe a
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difference in depth of responding neurons comparing either ipsi- to contralateral side or healthy and
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injured animals. Spikes of the same shape and amplitude were considered to reflect the activity of a single unit. Responsiveness to versatile stimuli and not only to painful, high threshold or low threshold stimuli suggests the activity of neurons integrating signals from different primary afferent sensory neurons (i.e. WDR neurons) in contrast to neurons mainly responding to one stimulus modality such as nociceptive-specific neurons in the spinal cord. In the group of mice studied, no spontaneous activity of
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the WDR neurons was observed. All neurons responding to an area of the plantar surface of the hind paw were included in the statistical analysis. Electrical signals were amplified (DAM80, WPI, U.S.A.), digitized (1408, CED, Cambridge, UK), displayed on a computer screen and analysed by Spike2
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software (CED, Cambridge, UK). Receptive field sizes were measured by mechanically stimulating
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every mm² of the skin with a mild stimulus (10 g von Frey, duration = 1 s) starting at the toes and proceeding proximally to the heel and beyond. A spot causing activity in the recorded neuron was defined as part of the receptive field. The stimulus interval was longer than the neuronal discharge duration and no temporal summation or wind-up was detected. For somatotopic mapping, on average, 16 neurons (8 on each side) were recorded from the same animal. The search for paw-innervating neurons continued until a neuron with receptive field of ankle or buttock was encountered on both the ipsi- as well as the contralateral side.
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ACCEPTED MANUSCRIPT Statistics Statistical data analysis was done with GraphPad Prism using student’s t-tests and ANOVA with posthoc multiple comparison testing if the distribution of the data showed no deviation from normality. If
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normality was violated Mann Whitney tests were performed. In case of multiple t-tests, consistent SD was not assumed among samples and the desired FDR (Q) was set to 1%. Graphs illustrate mean and
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SEM (whiskers) of a data set. Significance levels of p<0.01 are indicated by * unless otherwise noted.
Results
Development and resolution of bilateral posttraumatic changes in mechanical sensitivity and spinal
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neuronal receptive field size of fractured mice
Paw withdrawal thresholds to von Frey hair stimulations decreased four weeks after tibia fracture and
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recovered twelve weeks after the trauma. Interestingly, the contralateral healthy side also showed mechanical hypersensitivity compared to the naïve state without fracture (fig. 1A). Similarities were
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observed in the time course of receptive field size changes of spinal WDR neurons: four weeks after tibia fracture field sizes increased on both the ipsilateral as well as the contralateral side. Here, the mean size of the receptive fields (fig.1B) on the ipsilateral hind paw, mapped using low-, medium- and strong-intensity stimulation with 10, 26 and 80 g von Frey hair stimuli, respectively, was significantly increased in the post fractured group (µ=85 mm², ipsilateral) compared to uninjured animals (µ=20 mm²) and compared to the contralateral side of the same animal (µ=59 mm², contralateral).
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ACCEPTED MANUSCRIPT Signs of inflammation and pain Three weeks after tibia fracture we observed signs of posttraumatic inflammation in the mice: warming of the skin (fig. 2A), edema in the paw of the injured leg (fig. 2B) and a shift in weight bearing (fig. 2C)
inflammation observed on the contralateral paw.
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Activity characteristics of WDR neurons
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on the injured side. All these signs subsided twelve weeks after the trauma. There were no signs of
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Extracellular recordings from WDR neurons having receptive fields in the right fractured hind paw revealed discharge frequencies which increased with increasing stimulation strength (Fig. 3). Compared to healthy control animals, no significant difference in firing frequency of spinal neurons (n=28 each) was observed in mice suffering from tibia fracture with subsequent limb immobilization for three weeks.
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Recordings were done one week after cast removal.
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Caudal extension of the spinal paw representation area on the injured side To locate the whole sensory representation area of the hind paw in the dorsal horn of the thoracic/
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lumbar spinal cord, neurons were recorded in the region of the vertebrae Th11 to L1 (fig. 4A) and their receptive fields were mapped. The caudal and rostral borders of paw representations were delimited by locating neurons not responding to paw stimulations but adjacent regions of ankle, leg or buttock. Locations of these cells are depicted by a plus symbol (fig. 4B). Four weeks after bone fracture and casting, the rostral border did not differ between left and right side, but the caudal border was caudally shifted on the right, injured side (fig. 4B, arrow). The distribution of the receptive fields over the plantar surface of the paw was similar between study groups and sides of the body. Large fields with sizes over
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ACCEPTED MANUSCRIPT 100 mm², covering the whole foot and parts of the leg, occurred after bone fracture, mainly on the injured side (fig. 4C).
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Receptive fields of some spinal neurons increase after tibia fracture Receptive fields of spinal dorsal horn WDR neurons representing (parts of) the hind paw ranged from 245 mm² with similar means on both sides of 19 mm² for the left and 17 mm² for the right paw (fig. 5A).
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Four weeks after tibia fracture (fig. 5B), we found cells that displayed large fields (100-300 mm²). This portion of cells was neither seen in the uninjured group nor twelve weeks after the trauma. The
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enlargement of the field sizes occurred both on the injured and on the contralateral side. The portion of those cells with large receptive fields was greater on the injured side compared to the non-injured side.
Discussion
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After twelve weeks (fig. 5C), receptive fields ranged from 2-46 mm², similar to the uninjured state.
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The tibia fracture model was introduced to study behavioral, morphological and biochemical changes which occur in response to the fracture with three weeks casting. Our results on hypersensitivity, edema and pain are comparable to previous studies in rats20. Mechanical hypersensitivity can persist over a time span of several weeks in rats56. In the present study on mice mechanical hypersensitivity declined within 12 weeks, potentially due to species characteristics. Mice show signs of inflammation and posttraumatic pain which might be caused by a plethora of inflammatory signal molecules in the peripheral and central nervous system 5,23,32. We discovered comprehensive changes in the receptive field size of spinal dorsal horn neurons that accompany this posttraumatic stage, suggesting a major 11
ACCEPTED MANUSCRIPT reorganization of spinal circuitry after a trauma. This process was partially bilateral which nicely fits with subtle behavioral changes seen in previous experiments 18 and was reversible twelve weeks after fracture. After removing the cast, the mice demonstrated signs of inflammation and pain behavior as expected
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from studies on rats21 and recently also on mice 23. This demonstrates the validity of the mouse tibia fracture model for a posttraumatic inflammation. This posttraumatic reaction also occurs in humans after fracture and casting and is characterized by swelling, warming and pain, similar to an initiating CRPS.
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We can assume that “normal” posttraumatic inflammation and early CRPS might share some molecular
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mechanisms4,6 and that a posttraumatic reaction might constitute the very first pathophysiological changes which can initiate CRPS – with yet unknown causality. Processes transforming normal fracture healing into CRPS cannot presently be distinguished. The reaction of the rodents in our study presents as a “normal” posttraumatic inflammatory response which subsides over weeks but might – however – be the starting point of CRPS. Based on a robustly emerging posttraumatic inflammatory phenotype in
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all mice, we studied electrophysiological properties of spinal neurons to gain first insights into the spinal neuronal network functionality after a bone trauma and related pain and inflammation.
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The firing frequency of WDR neurons representing areas of the hind paw was not significantly changed after bone fracture. This is not an unusual finding in chronic pain because electrically evoked responses
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of dorsal horn neurons also did not change after peripheral nerve ligation in rats 41. Similarly, in the spinal nerve ligation model the characteristics of evoked spinal neuronal responses were comparable between the spinal nerve ligation and the control groups 47. Since spiking was unaltered in spinal neurons of fractured mice we suppose that central inhibition remains intact after bone fracture and the resolution of symptoms after 12 weeks might be due to a reversal of the peripheral changes. The amount and nature of spontaneous pain, a phenomenon that often but not always accompanies human CRPS8 was not studied in the animals because of a lack of appropriate tests52.
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ACCEPTED MANUSCRIPT The most noteworthy finding was the enlargement of receptive fields of spinal neurons four weeks after tibia fracture with subsequent immobilization. This enlargement occurred on both the ipsilateral and the contralateral side and fits to the hyperalgesia behavior of the mice during von Frey hair testing which was also partially bilateral. Thus, the enlargement of the receptive field sizes might reflect spinal
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reorganization and sensitization of the nociceptive pathways 24. This has been shown to occur in models of neuropathy 36,37 and other tissue damage models 27. Also in the partial sciatic nerve ligation (PNL) model of neuropathic pain, mechanical receptive fields of both ipsi- and contralateral dorsal horn
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neurons were found to be enlarged 51. In the PNL, enlargement of spinal receptive fields indicated a progressive central sensitization after 16 weeks. In the tibia fracture model central reorganization must
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occur much quicker since massive spinal changes were visible 4 weeks after the trauma. Furthermore, studies on neuropathic rats report smaller changes to the receptive field size than seen here 3. A trauma including inflammation may result in fast enlargement of spinal receptive fields as shown for the adjuvant-induced pain model25. It was recently shown for dorsal root ganglion neurons to align firing due
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to glial gap junctions during inflammation and thus contribute to pain hypersensitivity28. This suggests that the fracture with the posttraumatic inflammatory reaction plus the subsequent limb immobilization immediately initiates very extensive and complex disturbances of the spinal neuronal organization.
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Indeed, a fractured limb which has been immobilized responds to the trauma with stronger vascular, motor and sensory changes compared to a non-immobilized limb in men53 and mice 22. Thus an
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exaggerated reaction might partly be due to immobilization, which also applies to CRPS where non-use of the injured limb usually hampers recovery2. Interestingly, not all neurons changed their receptive field, as “normal” sizes ranging from 2-45 mm² were found in all groups. Four weeks after fracture, however, a portion of neurons occurred with huge fields spreading across large parts of the leg and back and with a significant caudal extension on the somatotopic map. Whether this was due to new formation of synapses 1 or strengthening of altered existing (silent) synapses 58 or different intrinsic firing properties of spinal neurons 43 cannot be 13
ACCEPTED MANUSCRIPT determined with this set of experiments. All three possibilities have been discussed extensively 9,15. In any case, our findings indicate the reorganization in the spinal sensory network due to the trauma and its treatment. The existence of multi-segmental projections of afferents that physiologically make functional but weak synaptic connections to dorsal horn neurons some millimeters rostral and caudal to
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the region where their receptive field is represented in the somatotopic map is well known 29. These multi-segmental connections might become strengthened after the trauma. Potentially, spinal cells that are connected to long-ranging afferents will enlarge their receptive field upon increased peripheral input
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due to limb trauma or due to spinal inflammatory changes 34.
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In healthy mice, as well as twelve weeks after the bone fracture, we found a somatotopic map that matches the vertebrate tetrapode spinal representations of extremities in the dorsal horn of the spinal cord 35. However, the representation of the paw caudally extended on the side of the injured extremity four weeks after tibia fracture. After a nerve lesion, this caudal extension develops after three weeks and persists 41. The tibia fracture model does not include anatomical denervation. This means that the
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transient caudal extension of the receptive fields is functional, e.g. by changes in the synaptic strength, than anatomical, e.g. by sprouting. This functional reorganization might also explain why most cells in the caudally extended region have large receptive fields (fig 4). The increase in receptive field sizes
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might also be associated with the changes seen in sensitivity behavior of the mice on both body sides.
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The caudal extension of the receptive fields could relate to changes in body perception and representation in the primary somatosensory cortex which has been shown for human CRPS or other immobilized patients39.
Dynamic receptive field plasticity in rodent spinal cord dorsal horn has been demonstrated after brief but strong input from nociceptive fibers 13. This transient change of receptive field sizes demonstrates the capability of the spinal network to adapt and recover. In contrast to the transient symptoms in the tibia fracture mouse model, untreated human CRPS usually worsens over time suggesting yet unknown factors that oppose the normal healing process after a trauma. 14
ACCEPTED MANUSCRIPT Tactile acuity is impaired in chronic pain conditions including CRPS 26,42. This was suggested to be a cortical phenomenon 30. It has been demonstrated in animal experiments that capsaicin-sensitive nociceptors provide an inhibitory control on cortical excitability of non-nociceptive somatosensory cortical neurons 11.Our data suggest that these alterations of cortical sensory encoding could be initiated
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by spinal reorganization. After short painful stimuli to the skin of healthy subjects, tactile acuity is
impaired only in a zone surrounding the stimulation site for some hours 19. The mechanism behind this localized hypaesthesia most likely is a presynaptic inhibition of low threshold mechanoreceptive input
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into non-nociceptive pathways of the spinal cord 38. As not all spinal sensory neurons showed an
mice twelve weeks after the trauma.
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enlargement of their receptive fields, these cells might be involved in the recovery process seen in the
Both, the ipsi- as well as the contralateral side to the trauma changed in mechanical sensitivity and receptive field size of spinal neurons after bone fracture. Subtle bilateral changes of sensitivity and hyperalgesia are not uncommon in human chronic pain 54. This means that calculating differences in
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mechanical sensitivity or other readouts between the ipsi- and contralateral side 50,56 might miss the bilateral effects reported here. Further studies should consider effects on both sides separately to
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address spinal neuroplasticity in chronic pain. The present study provides first electrophysiological insights into spinal neuronal changes that are initiated by bone fracture and subsequent immobilization
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of the injured limb in mice. The changes of spinal receptive fields and behavioral hypersensitivity share dynamics of emergence and decay.
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ACCEPTED MANUSCRIPT Acknowledgement The authors would like to thank A. Wehle and H. Danker for their commitment, Georg Otto for technical assistance, Prof. Maik Stüttgen for help in spike2 scripting, Cora Rebhorn and Myriam Herrnberger for
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logistics and Cheryl Ernest for proofreading the manuscript. Financial support was provided by the Hopp Foundation, Project 23016006; the DFG Bi 579/8-1; the EU FP7 ncRNAPA, project number 602133 and the workers compensation insurance BGW, Mainz (all to FB); and grant no. 97388102 IFF to SH. There
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is no conflict of interests for any of the authors.
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ACCEPTED MANUSCRIPT Figure legends
Figure 1. Ipsi- as well as contralateral changes of both mechanical sensitivity and receptive field size of spinal neurons
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develop after tibia fracture in all mice and decline within twelve weeks. A. Mechanical hypersensitivity, tested with von Frey hairs, is visible four weeks after tibia fracture on both extremities (n=29). Compared to the uninjured control state (n=49), both the healthy as well as the injured paw display reduced withdrawal thresholds, with an additional significant difference between both sides at the four week time point. Twelve weeks after fracture, withdrawal thresholds were not different from
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the uninjured control state (n=19). B. Four weeks after fracture, receptive fields of spinal neurons were enlarged on both sides (comparing ipsilateral at 0 and 4 weeks and contralateral at 0 and 4 weeks, n=12 and 19 animals, respectively) plus a
to non-injured sizes (n=10 animals).
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significant difference in healthy versus injured side at the four week time point. Twelve weeks after fracture, fields recovered
Figure 2. Signs of posttraumatic inflammation occur in fractured mice. A. Paw thickness increased in the injured paw three
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weeks after fracture (n=16) and returned to normal after twelve weeks. B. Skin temperature of the injured paw increased significantly three weeks after fracture (n=17). C. Guarding, i.e., shift in weight bearing of the injured leg as compared to the healthy side (100%), was seen three weeks after fracture with approximately 50 % weight put on the injured leg compared to
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the healthy leg (n=15).
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Figure 3. Action potential frequencies of WDR neurons during mild to strong mechanical stimulation of the receptive field. A. Example of an action potential trace evoked by a mild stimulus of 6 g von Frey hair (indicated by a grey bar, duration: 2 s). The action potential marked with an arrow is magnified on the right. B. Example of an action potential trace evoked by a strong stimulus of 80 g von Frey hair (indicated by a grey bar, duration: 2 s). The action potential marked with an arrow is magnified on the right. C. Firing frequency of WDR neurons during mild, medium and strong stimuli with receptive fields in the right hind paw of healthy mice (black bars, n=7 animals) and fractured mice (white bars, n=7 animals). Increasing stimulus strength gives rise to significantly increased discharge frequencies but comparing firing in mice with and without fracture did not reveal a significant difference (two-way ANOVA and Sidak’s multiple comparisons test).
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ACCEPTED MANUSCRIPT Figure 4. WDR neurons with receptive fields in the paw show different localizations in the spinal cord on the injured side. A. Somatotopic map of representation areas superimposed over the dorsal surface of the exposed spinal cord (region Th11 – L1). After injury (on right leg) the representation area of the paw extends caudally. Right of the dorsal spinal vessel (grey), the recording electrode can be seen as a black vertical line. B. Location of recorded neurons left and right of the dorsal vein (grey line) in healthy animals, after tibia fracture and after recovery (0, 4 and 12 weeks after tibia fracture). Circles represent
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neurons having receptive fields in the paw with size being color coded from light (2 mm²) to dark (>100 mm²). Plus symbols represent neurons with receptive fields outside the paw (buttock or ankle). Four weeks after fracture, neurons at locations which did not represent the paw on the healthy side became responsive on the injured side (arrow). C. Examples of receptive
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field extensions ranging from 3 mm² on the healthy side (light line) to 140 mm² on the injured side (dark line).
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Figure 5. Dynamic size of the receptive fields after trauma. A. Without injury, the receptive fields range from 2-45 mm² with a mean of approx. 18 mm² (n=10 animals). B. Four weeks after tibia fracture, fields range from 2-300 mm² with means of approx. 60 mm² on the healthy side and approx. 80 mm² on the injured side (n=12 animals). Cells with large fields (>50 mm²) were 14/61 (23%) on the healthy side and 21/58 (36%) on the injured side. C. Twelve weeks after tibia fracture, field sizes
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range from 2-46 mm² with means of 16 mm² on the healthy side and 20 mm² on the injured side (n=8 animals).
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ACCEPTED MANUSCRIPT 33. Li WW, Guo TZ, Shi X, Czirr E, Stan T, Sahbaie P, Wyss-Coray T, Kingery WS, Clark JD: Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome. PAIN 155:2377–2389, 2014 34. Li WW, Guo TZ, Shi X, Sun Y, Wei T, Clark DJ, Kingery WS: Substance P spinal signaling induces glial activation and nociceptive sensitization after fracture. Neuroscience 310:73–90, 2015
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ACCEPTED MANUSCRIPT 49. Tajerian M, Leu D, Zou Y, Sahbaie P, Li W, Khan H, Hsu V, Kingery W, Huang TT, Becerra L, Clark JD: Brain neuroplastic changes accompany anxiety and memory deficits in a model of complex regional pain syndrome. Anesthesiology 121:852–865, 2014 50. Tajerian M, Sahbaie P, Sun Y, Leu D, Yang HY, Li W, Huang TT, Kingery W, David Clark J: Sex differences in a Murine Model of Complex Regional Pain Syndrome. Neurobiology of learning and memory 123:100–109, 2015
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58. Wilson P, Kitchener PD: Plasticity of cutaneous primary afferent projections to the spinal dorsal horn. Prog Neurobiol 48:105–129, 1996
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ACCEPTED MANUSCRIPT Highlights: Receptive Fields are enlarged in the spinal cord after bone trauma in mice.
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The representation area of the injured body part in extended in the murine spinal cord.
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The spinal neuronal changes develop and subside together with the behavioral hypersensitivity.
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S1526-5900(16)30369-8
DOI:
10.1016/j.jpain.2016.12.010
Reference:
YJPAI 3349
To appear in:
Journal of Pain
Received Date: 8 November 2016 Revised Date:
6 December 2016
Accepted Date: 21 December 2016
Please cite this article as: Hirsch S, Ibrahim A, Krämer L, Escolano-Lozano F, Schlereth T, Birklein F, Spinal Reorganization after Bone Fracture in Mice, Journal of Pain (2017), doi: 10.1016/ j.jpain.2016.12.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Hirsch_manuscript_JP2016
Article type:
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Original article in the section “Neurology” Title:
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Spinal Reorganization after Bone Fracture in Mice
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Running Head:
Bone trauma causes massive but reversible changes in spinal circuitry. Authors: Dr. Silke Hirsch (corresponding author)
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Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1
Germany
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55131 Mainz
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Tel: +49 170 4728743
Email: [email protected]
Alaa Ibrahim
Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1 55131 Mainz Germany 1
ACCEPTED MANUSCRIPT Laura Krämer Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1 55131 Mainz
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Germany
Fabiola Escolano-Lozano
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Klinik und Poliklinik für Neurologie Unimedizin Mainz
55131 Mainz Germany
Dr. Tanja Schlereth
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Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1
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55131 Mainz Germany
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Langenbeckstr. 1
Prof. Dr. Frank Birklein
Klinik und Poliklinik für Neurologie Unimedizin Mainz Langenbeckstr. 1 55131 Mainz Germany
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ACCEPTED MANUSCRIPT Disclosures: Research funding for this project was provided by the Hopp Foundation, Project 23016006; the DFG, Bi 579/8-1; the EU, FP7 ncRNAPA, project number 602133; the workers compensation insurance BGW
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Mainz (all to FB) and grant no. 97388102 IFF (for SH). There is no conflict of interests for any of the authors. All authors discussed the results and commented on the manuscript.
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Highlights:
Receptive fields are enlarged in the spinal cord after bone trauma in mice.
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The representation area of the injured body part is extended in the murine spinal cord.
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The spinal neuronal changes develop and subside together with the behavioral hypersensitivity.
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Abstract
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Bone fracture with subsequent immobilization of the injured limb can cause Complex Regional Pain
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Syndrome (CRPS) in humans. Mechanisms of CRPS are still not completely understood but bone fracture with casting in mice leads to a similar posttraumatic inflammation as seen in humans and might
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therefore be an analogue to human CRPS. Here we report behavioral and spinal electrophysiological changes in mice that developed swelling of the paw, warming of the skin and pain in the injured limb after bone fracture. The receptive field sizes of spinal neurons representing areas of the hind paws increased after trauma and recovered over time – as did the behavioral signs of inflammation and pain. Interestingly, both sides – the ipsi- and the contralateral limb – showed changes in mechanical sensitivity and neuronal network organization after the trauma. The characteristics of evoked neuronal responses recorded in the dorsal horn of the mice were similar between uninjured controls and fractured animals. However, we saw a caudal extension of the represented area of the hind paw in the spinal cord 3
ACCEPTED MANUSCRIPT at the injured side and an occurrence of large receptive fields of wide dynamic range neurons. The findings in mice compare to human symptoms in CRPS with ipsi- but also contralateral allodynia and pain. In all mice tested, all signs subsided twelve weeks after trauma. Our data suggest a significant
of neuropathies. This process seems to be reversible in the rodent.
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Perspective
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reorganization of spinal circuitry after limb trauma, in a degree more comprehensive than most models
The discovery of enlarged spinal neuronal receptive fields and caudal extension of the representation
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area of the injured body part which subsides several weeks after a bone trauma in mice might give hope to patients of Complex Regional Pain Syndrome if – in the future – we are able to translate the rodent
CRPS
Mouse
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Spinal cord
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Keywords
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recovery mechanisms to posttraumatic humans.
Receptive field
Hypersensitivity
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ACCEPTED MANUSCRIPT Introduction Bone fracture with subsequent immobilization of the injured limb can rarely cause a debilitating, painful syndrome called Complex Regional Pain Syndrome (CRPS) in human patients 40. CRPS is
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characterized by symptoms which go beyond a nerve innervation territory and spread distally. These symptoms are pain and hyperalgesia, signs of inflammation such as skin temperature changes and edema, motor disturbances and trophic changes. The full clinical picture of CRPS seems unique to humans, mainly because of a strong central pathophysiology component which is characterized by brain
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reorganization, body perception disturbances and learned maladaptive behavior. However, mammals
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like mice or rats develop a painful inflammatory phenotype as posttraumatic reaction after a bone fracture which is similar to humans 7. Thus, the tibia fracture mouse model 18,23 is employed here to study posttraumatic changes in behavior and spinal electrophysiology. This model shares posttraumatic inflammatory signs and neuropathic symptoms such as hypersensitivity to mild non-noxious stimuli 45,46 with human CRPS 40. These signs resemble the early inflammatory stage of CRPS and accordingly an
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increase of pro-inflammatory markers such as TNFα, NGF and Il-6 44,57 was found in rat and mouse skin, which has been found in skin biopsies from human CRPS patients as well 5. In contrast to human CRPS, symptoms occur almost regularly and disappear after a few weeks perhaps because of the
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lacking maladaptive learning of rodents.
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The pathophysiology leading to the phenotype in the tibia fracture mouse model is characterized by a massive immune response causing hyperalgesia 23 which is probably mediated by sensitization of primary afferent nociceptors like in other pain models 31 or second order afferent neurons in the spinal cord 45. The neuronal mechanisms underlying the hyperalgesia behavior in fractured mice are not yet specified. Receptive field reorganization is a characteristic of neuronal plasticity after peripheral nerve injury 16,17 and enlargement of receptive field sizes in response to low intensity mechanical stimuli has been suggested as an explanation for the development of allodynia 47. Enlarged receptive fields of spinal 5
ACCEPTED MANUSCRIPT neurons have been described in the chronic constriction injury model 14, after partial sciatic nerve ligation 51 and spinal nerve ligation 47. We therefore hypothesized that one neuronal signature of pain and hyperalgesia behavior of mice with
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distal tibia fracture and cast immobilization of the leg for three weeks may be the enlargement of receptive fields of those spinal neurons that receive afferent input from the fractured limb.
Comprehensive reorganization processes in the dorsal horn have been reported in a rat model of
cancer-induced bone pain 55. With our electrophysiological study we aim to estimate the extent of this
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reorganization and whether this spinal reorganization relates to hyperalgesia behavior.
Material and Methods Mice
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Experiments were conducted with 150 male mice, C57/bl6j. Animals were housed in group cages, food and water ad libitum, twelve hour dark/light cycle, in the institutional animal facilities. All procedures followed the guidelines for animal research of the German Animal Welfare Act 2006 and adhered to the
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guidelines of the Committee for Research and Ethical Issues of IASP 1983. They were approved by the German federal government and supervised by the institutional veterinarian. Two of the animals died
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before the end of experiments; the remaining 148 animals were included in the analysis. Electrophysiological experiments were conducted as the final experiment in the same behaviorally examined animals in order to reduce the number of animals needed. Group sizes were minimized according to precedent power analyses and suffering of animals was reduced to the minimum possible. Experimenters were blinded to the group of investigation. We adhered in sex and age to previous work on the tibia fracture model to make our study comparable to the literature – disregarding the fact that mechanisms might differ between males and females10.
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ACCEPTED MANUSCRIPT Bone Fracture At the age of 6 weeks, mice received a closed tibia fracture of the right leg with subsequent casting for 3 weeks. Surgery was done under isoflurane anaesthesia (2%) combined with buprenorphine analgesia
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during, 12 and 24 hours after surgery (0.05 mg/kg, s.c. each) and antibiosis (enrofloxacin, 5 mg/kg s.c.). To achieve a closed fracture of the shank without rupturing the skin, the leg was tightly held between two fingers; a swab was placed around the lower leg and held in place with an open needle holder. The needle holder was then bent so that the tibia would break once at the spot where the inner edge of the
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needle holder pressed against the swab. The pressure was released immediately after the audible click
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of the bone breaking was heard; the leg was returned to its straight position. In this physiological position the cast was placed around the lower leg, the trunk and the palm of the foot to support the ankle in a 90° position. After 3 weeks, casts were removed under isoflurane anaesthesia.
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Mechanical allodynia, weight bearing, edema and skin temperature measurement Mechanical allodynia was measured with von Frey hairs (Aesthesio, Ugo Basile) using an up-down
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paradigm 12 as previously used for sensitivity testing of bone fractured rodents 20,21. Weight bearing was measured with an incapacitance meter (IITC Life Science) following a protocol also established for
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fractured rodents 21,44. In brief, each hind limb was rested on a separate scale plate measuring the weight put on this leg for 5 s. With an interval of 10 s this measurement was repeated six times. Values were averaged. The incapacitance device has been validated by the group of W. Kingery, Palo Alto, in the mouse fracture model 23,33,48,49. Thickness of the hind paw was measured with a Laser sensor (4381 PreciCura, Limab, Sweden). Skin temperature of the hind paw was measured with a small wire thermometer (OM-CP, Omega, USA) which was placed onto the skin in between the toes 20,21.
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ACCEPTED MANUSCRIPT Electrophysiology A laminectomy was performed under isoflurane anaesthesia (2% during surgery, 1% during recording) to enable extracellular recordings of the activity of single wide dynamic range (WDR) neurons. The
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spinal cord under the vertebrae Th12-L1 was exposed to access the spinal segments innervating the hind paw. Tungsten electrodes (2 MOhm) were inserted into the deep (100-400 µm from dorsal surface) dorsal horn to record identified WDR neurons sorted by spike amplitude and characterized by their responsiveness to mild (6 g), medium (26 g) and strong (80 g) von Frey stimuli. We did not observe a
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difference in depth of responding neurons comparing either ipsi- to contralateral side or healthy and
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injured animals. Spikes of the same shape and amplitude were considered to reflect the activity of a single unit. Responsiveness to versatile stimuli and not only to painful, high threshold or low threshold stimuli suggests the activity of neurons integrating signals from different primary afferent sensory neurons (i.e. WDR neurons) in contrast to neurons mainly responding to one stimulus modality such as nociceptive-specific neurons in the spinal cord. In the group of mice studied, no spontaneous activity of
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the WDR neurons was observed. All neurons responding to an area of the plantar surface of the hind paw were included in the statistical analysis. Electrical signals were amplified (DAM80, WPI, U.S.A.), digitized (1408, CED, Cambridge, UK), displayed on a computer screen and analysed by Spike2
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software (CED, Cambridge, UK). Receptive field sizes were measured by mechanically stimulating
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every mm² of the skin with a mild stimulus (10 g von Frey, duration = 1 s) starting at the toes and proceeding proximally to the heel and beyond. A spot causing activity in the recorded neuron was defined as part of the receptive field. The stimulus interval was longer than the neuronal discharge duration and no temporal summation or wind-up was detected. For somatotopic mapping, on average, 16 neurons (8 on each side) were recorded from the same animal. The search for paw-innervating neurons continued until a neuron with receptive field of ankle or buttock was encountered on both the ipsi- as well as the contralateral side.
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ACCEPTED MANUSCRIPT Statistics Statistical data analysis was done with GraphPad Prism using student’s t-tests and ANOVA with posthoc multiple comparison testing if the distribution of the data showed no deviation from normality. If
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normality was violated Mann Whitney tests were performed. In case of multiple t-tests, consistent SD was not assumed among samples and the desired FDR (Q) was set to 1%. Graphs illustrate mean and
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SEM (whiskers) of a data set. Significance levels of p<0.01 are indicated by * unless otherwise noted.
Results
Development and resolution of bilateral posttraumatic changes in mechanical sensitivity and spinal
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neuronal receptive field size of fractured mice
Paw withdrawal thresholds to von Frey hair stimulations decreased four weeks after tibia fracture and
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recovered twelve weeks after the trauma. Interestingly, the contralateral healthy side also showed mechanical hypersensitivity compared to the naïve state without fracture (fig. 1A). Similarities were
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observed in the time course of receptive field size changes of spinal WDR neurons: four weeks after tibia fracture field sizes increased on both the ipsilateral as well as the contralateral side. Here, the mean size of the receptive fields (fig.1B) on the ipsilateral hind paw, mapped using low-, medium- and strong-intensity stimulation with 10, 26 and 80 g von Frey hair stimuli, respectively, was significantly increased in the post fractured group (µ=85 mm², ipsilateral) compared to uninjured animals (µ=20 mm²) and compared to the contralateral side of the same animal (µ=59 mm², contralateral).
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ACCEPTED MANUSCRIPT Signs of inflammation and pain Three weeks after tibia fracture we observed signs of posttraumatic inflammation in the mice: warming of the skin (fig. 2A), edema in the paw of the injured leg (fig. 2B) and a shift in weight bearing (fig. 2C)
inflammation observed on the contralateral paw.
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Activity characteristics of WDR neurons
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on the injured side. All these signs subsided twelve weeks after the trauma. There were no signs of
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Extracellular recordings from WDR neurons having receptive fields in the right fractured hind paw revealed discharge frequencies which increased with increasing stimulation strength (Fig. 3). Compared to healthy control animals, no significant difference in firing frequency of spinal neurons (n=28 each) was observed in mice suffering from tibia fracture with subsequent limb immobilization for three weeks.
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Recordings were done one week after cast removal.
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Caudal extension of the spinal paw representation area on the injured side To locate the whole sensory representation area of the hind paw in the dorsal horn of the thoracic/
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lumbar spinal cord, neurons were recorded in the region of the vertebrae Th11 to L1 (fig. 4A) and their receptive fields were mapped. The caudal and rostral borders of paw representations were delimited by locating neurons not responding to paw stimulations but adjacent regions of ankle, leg or buttock. Locations of these cells are depicted by a plus symbol (fig. 4B). Four weeks after bone fracture and casting, the rostral border did not differ between left and right side, but the caudal border was caudally shifted on the right, injured side (fig. 4B, arrow). The distribution of the receptive fields over the plantar surface of the paw was similar between study groups and sides of the body. Large fields with sizes over
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ACCEPTED MANUSCRIPT 100 mm², covering the whole foot and parts of the leg, occurred after bone fracture, mainly on the injured side (fig. 4C).
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Receptive fields of some spinal neurons increase after tibia fracture Receptive fields of spinal dorsal horn WDR neurons representing (parts of) the hind paw ranged from 245 mm² with similar means on both sides of 19 mm² for the left and 17 mm² for the right paw (fig. 5A).
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Four weeks after tibia fracture (fig. 5B), we found cells that displayed large fields (100-300 mm²). This portion of cells was neither seen in the uninjured group nor twelve weeks after the trauma. The
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enlargement of the field sizes occurred both on the injured and on the contralateral side. The portion of those cells with large receptive fields was greater on the injured side compared to the non-injured side.
Discussion
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After twelve weeks (fig. 5C), receptive fields ranged from 2-46 mm², similar to the uninjured state.
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The tibia fracture model was introduced to study behavioral, morphological and biochemical changes which occur in response to the fracture with three weeks casting. Our results on hypersensitivity, edema and pain are comparable to previous studies in rats20. Mechanical hypersensitivity can persist over a time span of several weeks in rats56. In the present study on mice mechanical hypersensitivity declined within 12 weeks, potentially due to species characteristics. Mice show signs of inflammation and posttraumatic pain which might be caused by a plethora of inflammatory signal molecules in the peripheral and central nervous system 5,23,32. We discovered comprehensive changes in the receptive field size of spinal dorsal horn neurons that accompany this posttraumatic stage, suggesting a major 11
ACCEPTED MANUSCRIPT reorganization of spinal circuitry after a trauma. This process was partially bilateral which nicely fits with subtle behavioral changes seen in previous experiments 18 and was reversible twelve weeks after fracture. After removing the cast, the mice demonstrated signs of inflammation and pain behavior as expected
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from studies on rats21 and recently also on mice 23. This demonstrates the validity of the mouse tibia fracture model for a posttraumatic inflammation. This posttraumatic reaction also occurs in humans after fracture and casting and is characterized by swelling, warming and pain, similar to an initiating CRPS.
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We can assume that “normal” posttraumatic inflammation and early CRPS might share some molecular
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mechanisms4,6 and that a posttraumatic reaction might constitute the very first pathophysiological changes which can initiate CRPS – with yet unknown causality. Processes transforming normal fracture healing into CRPS cannot presently be distinguished. The reaction of the rodents in our study presents as a “normal” posttraumatic inflammatory response which subsides over weeks but might – however – be the starting point of CRPS. Based on a robustly emerging posttraumatic inflammatory phenotype in
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all mice, we studied electrophysiological properties of spinal neurons to gain first insights into the spinal neuronal network functionality after a bone trauma and related pain and inflammation.
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The firing frequency of WDR neurons representing areas of the hind paw was not significantly changed after bone fracture. This is not an unusual finding in chronic pain because electrically evoked responses
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of dorsal horn neurons also did not change after peripheral nerve ligation in rats 41. Similarly, in the spinal nerve ligation model the characteristics of evoked spinal neuronal responses were comparable between the spinal nerve ligation and the control groups 47. Since spiking was unaltered in spinal neurons of fractured mice we suppose that central inhibition remains intact after bone fracture and the resolution of symptoms after 12 weeks might be due to a reversal of the peripheral changes. The amount and nature of spontaneous pain, a phenomenon that often but not always accompanies human CRPS8 was not studied in the animals because of a lack of appropriate tests52.
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ACCEPTED MANUSCRIPT The most noteworthy finding was the enlargement of receptive fields of spinal neurons four weeks after tibia fracture with subsequent immobilization. This enlargement occurred on both the ipsilateral and the contralateral side and fits to the hyperalgesia behavior of the mice during von Frey hair testing which was also partially bilateral. Thus, the enlargement of the receptive field sizes might reflect spinal
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reorganization and sensitization of the nociceptive pathways 24. This has been shown to occur in models of neuropathy 36,37 and other tissue damage models 27. Also in the partial sciatic nerve ligation (PNL) model of neuropathic pain, mechanical receptive fields of both ipsi- and contralateral dorsal horn
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neurons were found to be enlarged 51. In the PNL, enlargement of spinal receptive fields indicated a progressive central sensitization after 16 weeks. In the tibia fracture model central reorganization must
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occur much quicker since massive spinal changes were visible 4 weeks after the trauma. Furthermore, studies on neuropathic rats report smaller changes to the receptive field size than seen here 3. A trauma including inflammation may result in fast enlargement of spinal receptive fields as shown for the adjuvant-induced pain model25. It was recently shown for dorsal root ganglion neurons to align firing due
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to glial gap junctions during inflammation and thus contribute to pain hypersensitivity28. This suggests that the fracture with the posttraumatic inflammatory reaction plus the subsequent limb immobilization immediately initiates very extensive and complex disturbances of the spinal neuronal organization.
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Indeed, a fractured limb which has been immobilized responds to the trauma with stronger vascular, motor and sensory changes compared to a non-immobilized limb in men53 and mice 22. Thus an
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exaggerated reaction might partly be due to immobilization, which also applies to CRPS where non-use of the injured limb usually hampers recovery2. Interestingly, not all neurons changed their receptive field, as “normal” sizes ranging from 2-45 mm² were found in all groups. Four weeks after fracture, however, a portion of neurons occurred with huge fields spreading across large parts of the leg and back and with a significant caudal extension on the somatotopic map. Whether this was due to new formation of synapses 1 or strengthening of altered existing (silent) synapses 58 or different intrinsic firing properties of spinal neurons 43 cannot be 13
ACCEPTED MANUSCRIPT determined with this set of experiments. All three possibilities have been discussed extensively 9,15. In any case, our findings indicate the reorganization in the spinal sensory network due to the trauma and its treatment. The existence of multi-segmental projections of afferents that physiologically make functional but weak synaptic connections to dorsal horn neurons some millimeters rostral and caudal to
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the region where their receptive field is represented in the somatotopic map is well known 29. These multi-segmental connections might become strengthened after the trauma. Potentially, spinal cells that are connected to long-ranging afferents will enlarge their receptive field upon increased peripheral input
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due to limb trauma or due to spinal inflammatory changes 34.
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In healthy mice, as well as twelve weeks after the bone fracture, we found a somatotopic map that matches the vertebrate tetrapode spinal representations of extremities in the dorsal horn of the spinal cord 35. However, the representation of the paw caudally extended on the side of the injured extremity four weeks after tibia fracture. After a nerve lesion, this caudal extension develops after three weeks and persists 41. The tibia fracture model does not include anatomical denervation. This means that the
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transient caudal extension of the receptive fields is functional, e.g. by changes in the synaptic strength, than anatomical, e.g. by sprouting. This functional reorganization might also explain why most cells in the caudally extended region have large receptive fields (fig 4). The increase in receptive field sizes
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might also be associated with the changes seen in sensitivity behavior of the mice on both body sides.
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The caudal extension of the receptive fields could relate to changes in body perception and representation in the primary somatosensory cortex which has been shown for human CRPS or other immobilized patients39.
Dynamic receptive field plasticity in rodent spinal cord dorsal horn has been demonstrated after brief but strong input from nociceptive fibers 13. This transient change of receptive field sizes demonstrates the capability of the spinal network to adapt and recover. In contrast to the transient symptoms in the tibia fracture mouse model, untreated human CRPS usually worsens over time suggesting yet unknown factors that oppose the normal healing process after a trauma. 14
ACCEPTED MANUSCRIPT Tactile acuity is impaired in chronic pain conditions including CRPS 26,42. This was suggested to be a cortical phenomenon 30. It has been demonstrated in animal experiments that capsaicin-sensitive nociceptors provide an inhibitory control on cortical excitability of non-nociceptive somatosensory cortical neurons 11.Our data suggest that these alterations of cortical sensory encoding could be initiated
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by spinal reorganization. After short painful stimuli to the skin of healthy subjects, tactile acuity is
impaired only in a zone surrounding the stimulation site for some hours 19. The mechanism behind this localized hypaesthesia most likely is a presynaptic inhibition of low threshold mechanoreceptive input
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into non-nociceptive pathways of the spinal cord 38. As not all spinal sensory neurons showed an
mice twelve weeks after the trauma.
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enlargement of their receptive fields, these cells might be involved in the recovery process seen in the
Both, the ipsi- as well as the contralateral side to the trauma changed in mechanical sensitivity and receptive field size of spinal neurons after bone fracture. Subtle bilateral changes of sensitivity and hyperalgesia are not uncommon in human chronic pain 54. This means that calculating differences in
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mechanical sensitivity or other readouts between the ipsi- and contralateral side 50,56 might miss the bilateral effects reported here. Further studies should consider effects on both sides separately to
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address spinal neuroplasticity in chronic pain. The present study provides first electrophysiological insights into spinal neuronal changes that are initiated by bone fracture and subsequent immobilization
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of the injured limb in mice. The changes of spinal receptive fields and behavioral hypersensitivity share dynamics of emergence and decay.
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ACCEPTED MANUSCRIPT Acknowledgement The authors would like to thank A. Wehle and H. Danker for their commitment, Georg Otto for technical assistance, Prof. Maik Stüttgen for help in spike2 scripting, Cora Rebhorn and Myriam Herrnberger for
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logistics and Cheryl Ernest for proofreading the manuscript. Financial support was provided by the Hopp Foundation, Project 23016006; the DFG Bi 579/8-1; the EU FP7 ncRNAPA, project number 602133 and the workers compensation insurance BGW, Mainz (all to FB); and grant no. 97388102 IFF to SH. There
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is no conflict of interests for any of the authors.
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Figure 1. Ipsi- as well as contralateral changes of both mechanical sensitivity and receptive field size of spinal neurons
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develop after tibia fracture in all mice and decline within twelve weeks. A. Mechanical hypersensitivity, tested with von Frey hairs, is visible four weeks after tibia fracture on both extremities (n=29). Compared to the uninjured control state (n=49), both the healthy as well as the injured paw display reduced withdrawal thresholds, with an additional significant difference between both sides at the four week time point. Twelve weeks after fracture, withdrawal thresholds were not different from
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the uninjured control state (n=19). B. Four weeks after fracture, receptive fields of spinal neurons were enlarged on both sides (comparing ipsilateral at 0 and 4 weeks and contralateral at 0 and 4 weeks, n=12 and 19 animals, respectively) plus a
to non-injured sizes (n=10 animals).
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significant difference in healthy versus injured side at the four week time point. Twelve weeks after fracture, fields recovered
Figure 2. Signs of posttraumatic inflammation occur in fractured mice. A. Paw thickness increased in the injured paw three
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weeks after fracture (n=16) and returned to normal after twelve weeks. B. Skin temperature of the injured paw increased significantly three weeks after fracture (n=17). C. Guarding, i.e., shift in weight bearing of the injured leg as compared to the healthy side (100%), was seen three weeks after fracture with approximately 50 % weight put on the injured leg compared to
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the healthy leg (n=15).
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Figure 3. Action potential frequencies of WDR neurons during mild to strong mechanical stimulation of the receptive field. A. Example of an action potential trace evoked by a mild stimulus of 6 g von Frey hair (indicated by a grey bar, duration: 2 s). The action potential marked with an arrow is magnified on the right. B. Example of an action potential trace evoked by a strong stimulus of 80 g von Frey hair (indicated by a grey bar, duration: 2 s). The action potential marked with an arrow is magnified on the right. C. Firing frequency of WDR neurons during mild, medium and strong stimuli with receptive fields in the right hind paw of healthy mice (black bars, n=7 animals) and fractured mice (white bars, n=7 animals). Increasing stimulus strength gives rise to significantly increased discharge frequencies but comparing firing in mice with and without fracture did not reveal a significant difference (two-way ANOVA and Sidak’s multiple comparisons test).
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ACCEPTED MANUSCRIPT Figure 4. WDR neurons with receptive fields in the paw show different localizations in the spinal cord on the injured side. A. Somatotopic map of representation areas superimposed over the dorsal surface of the exposed spinal cord (region Th11 – L1). After injury (on right leg) the representation area of the paw extends caudally. Right of the dorsal spinal vessel (grey), the recording electrode can be seen as a black vertical line. B. Location of recorded neurons left and right of the dorsal vein (grey line) in healthy animals, after tibia fracture and after recovery (0, 4 and 12 weeks after tibia fracture). Circles represent
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neurons having receptive fields in the paw with size being color coded from light (2 mm²) to dark (>100 mm²). Plus symbols represent neurons with receptive fields outside the paw (buttock or ankle). Four weeks after fracture, neurons at locations which did not represent the paw on the healthy side became responsive on the injured side (arrow). C. Examples of receptive
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field extensions ranging from 3 mm² on the healthy side (light line) to 140 mm² on the injured side (dark line).
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Figure 5. Dynamic size of the receptive fields after trauma. A. Without injury, the receptive fields range from 2-45 mm² with a mean of approx. 18 mm² (n=10 animals). B. Four weeks after tibia fracture, fields range from 2-300 mm² with means of approx. 60 mm² on the healthy side and approx. 80 mm² on the injured side (n=12 animals). Cells with large fields (>50 mm²) were 14/61 (23%) on the healthy side and 21/58 (36%) on the injured side. C. Twelve weeks after tibia fracture, field sizes
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range from 2-46 mm² with means of 16 mm² on the healthy side and 20 mm² on the injured side (n=8 animals).
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ACCEPTED MANUSCRIPT Highlights: Receptive Fields are enlarged in the spinal cord after bone trauma in mice.
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The representation area of the injured body part in extended in the murine spinal cord.
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The spinal neuronal changes develop and subside together with the behavioral hypersensitivity.
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