Elsevier

Neuroscience Letters

Volume 739, 20 November 2020, 135429
Neuroscience Letters

Spinal caspase-3 contributes to tibial fracture-associated postoperative allodynia via up-regulation of LRRTM1 expression in mice

https://doi.org/10.1016/j.neulet.2020.135429Get rights and content

Highlights

  • Tibial fracture induces mechanical and cold allodynia after surgery.

  • Tibial fracture increases spinal caspase-3 activity and LRRTM1 expression.

  • Spinal caspase-3 inhibition reduces fracture-associated postoperative allodynia and LRRTM1 expression.

  • LRRTM1 knockdown alleviates fracture-associated postoperative allodynia.

  • LRRTM1 knockdown reverses the caspase-3 evoked acute pain.

Abstract

Background

Bone fracture may subsequently cause chronic postoperative pain after orthopedic surgery, but mechanisms remain elusive. The necessity of caspase-3 in neuroinflammation and synaptic plasticity has been summarized in pathological pain. Leucine-rich repeat transmembrane protein 1 (LRRTM1) mediates synaptic delivery of AMPA receptor and synaptogenesis. This study evaluated whether caspase-3 and LRRTM1 are required for fracture-associated postoperative allodynia.

Methods

A model of tibial fracture with intramedullary pinning in mice was established for the induction of postoperative pain, verified by measurement of mechanical paw withdrawal threshold and cold scores response to acetone. The caspase-3 specific inhibitor, recombinant caspase-3 and LRRTM1 knockdown by shRNA were utilized for the investigation of pathogenesis as well as the prevention of allodynia. Also, the activity of caspase-3 and the expression of LRRTM1 in the spinal dorsal horn were examined by Western blot and RT-qPCR.

Results

This study reported that tibial fracture and orthopedic surgery produced long-lasting mechanical allodynia and cold allodynia, along with the up-modulation of spinal caspase-3 activity (but not caspase-3 expression) and LRRTM1 expression. Spinal caspase-3 inhibition prevented fracture-associated behavioral allodynia in a dose-dependent manner. Caspase-3 inhibitor also reduced the spinal increased LRRTM1 level after tibial fracture with pinning. Spinal LRRTM1 deficiency impaired fracture-caused postoperative pain. Intrathecal recombinant caspase-3 facilitated acute pain hypersensitivity and spinal LRRTM1 expression in naïve mice, reversing by LRRTM1 knockdown.

Conclusion

Our current results demonstrate the spinal up-regulation of LRRTM1 by caspase-3 activation in the development of tibial fracture-associated postoperative pain in mice.

Introduction

Chronic postoperative pain after orthopedic injuries and subsequent surgery continues to be a serious clinical problem which hinders the physical rehabilitation and burdens healthcare systems worldwide [1]. Also, current existing treatments of non-steroidal anti-inflammatory drugs and opioids may potentially impair bone healing [2,3]. Neuroinflammation, neural excitability and synaptic plasticity are considered as positive effectors in the development of chronic pathologic pain after fracture and orthopedic repairs [[4], [5], [6], [7]], although underlying pathophysiology remains to be virtually elucidated.

The majority of investigations have recapitulated that caspases, intracellular cysteine proteases, are responsible for apoptosis and neurodegeneration [8]. Recently, several caspases are reported in inflammatory pain, neuropathic pain, paclitaxel-induced allodynia and remifentanil-induced hyperalgesia via facilitating the release of inflammatory mediators and the transmission of excitatory synapses [[9], [10], [11], [12]]. Among all caspases, caspase-3 is increased in a model of bone cancer pain in rodents, simultaneously, spinal caspase-3 knockdown alleviates neuropathic pain after peripheral nerve injury [13,14]. More importantly, the contribution of caspase-3 in α-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated neuronal excitotoxicity has been identified [15]. Given an important role of AMPAR in chronic pain-related syndromes [4,[16], [17], [18]], we investigated whether and how spinal caspase-3 mediates fracture-associated postoperative pain.

Leucine-rich repeat transmembrane proteins (LRRTMs) are synaptic cell adhesion molecules that drive excitatory synapse formation as well as affect synaptic function [19,20]. LRRTM1 has been recognized for its prominent role in the maintenance of AMPAR density at synapses and long-term potentiation (LTP) [21,22]. Also, LRRTM1 deletion in adult mice impairs the synaptosomal insertion of AMPAR and AMPAR-mediated synaptic transmission in hippocampal neurons [23]. However, it remains fully unclear whether LRRTM1 is required for fracture-associated postoperative pain.

In this present study, we characterized the substantial interaction between caspase-3 and LRRTM1 in postoperative allodynia caused by tibial fracture and orthopedic repairs. Specifically, spinal caspase-3 activation and LRRTM1 expression were determined. The pharmacologic caspase-3 inhibition and LRRTM1 knockdown with intrathecal shRNA confirmed the mechanism and prevention of pain phenotypes. We herein summarized that targeting caspase-3/LRRTM1 signaling might be beneficial for the therapeutic intervention in fracture-associated postoperative pain condition.

Section snippets

Animals

Male C57BL/6 mice (Adult, 8–12 weeks) were supplied by the Laboratory Animal Center of the Military Medical Science Academy of the Chinese People’s Liberation Army. All mice were housed (< 5 mice/cage) in a temperature-controlled (22−25 °C) room under a 12 h light/dark cycles and fed a standard diet and water. The care and treatment of experimental animals conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals. All animal procedures were approved by the

The induction and maintenance of mechanical allodynia and cold allodynia after tibial fracture and orthopedic surgery

First, no significant differences in the basal mechanical and cold sensitivity was detected between two groups (P >  0.05, n = 6, Fig. 1A and B). Sham animals exhibited a mild decrease in the paw withdrawal threshold on day 3 after surgery as compared to baseline (P < 0.05, Fig. 1A), suggesting the production of transient mechanical allodynia after sham operation. The acetone test revealed a slight increase in cold response scores for 3 days in sham mice (P < 0.05, Fig. 1B), suggesting the

Discussion

The present study identifies the contribution of spinal caspase-3 activation in the expression of LRRTM1 pathway after tibial fracture and orthopedic surgery, which in turn underlies the pathogenesis of fracture-associated postoperative pain phenotype. Specifically, mechanical allodynia and cold allodynia are evident from day 3 to at least day 21 during the post-surgical period, which is accompanied by up-modulation of caspase-3 activity and LRRTM1 expression in the spinal dorsal horn.

CRediT authorship contribution statement

Linlin Zhang: Data curation, Project administration, Writing - original draft, Funding acquisition. Jing Li: Visualization, Investigation, Validation. Yize Li: Visualization, Investigation, Validation. Zhen Wang: Formal analysis, Methodology, Software, Validation. Guolin Wang: Conceptualization, Writing - review & editing. Yonghao Yu: Visualization, Investigation. Chengcheng Song: Visualization, Investigation, Writing - review & editing. Wei Cui: Supervision, Conceptualization, Writing - review

Declaration of Competing Interest

The authors have declared that no conflict of interest exists.

Acknowledgments

This work was supported by research grants from the National Natural Science Foundation of China (81801107, 81571077, 81500961, and 81400908).

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