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