The mechanisms underlying CPSS remain partly understood, and there is a dearth of an animal model miming clinical CPSS conditions. Here, we utilized rats initially subjected to rod insertion to induce LBP, followed by a decompression surgery with rod removal and laminectomy, as a potential clinically pertinent CPSS model. Moreover, we showed that systemic administration of DALDA exhibited efficacy in ameliorating both mechanical and thermal hypersensitivity and attenuated the increased DRG neuron excitability in CPSS rats.

Although not statistically significant, there was a trend of reduced pain in rats that received rod removal and laminectomy (CPSS, Group 1) compared to rats with chronic rod retention (LBP, Group 3). Nevertheless, both CPSS and LBP mice developed long-lasting hypersensitivity to mechanical and thermal stimuli in the ipsilateral hind paw, aligning with the pain symptoms observed in these patients where individuals may experience heightened sensitivity to stimuli along one or both legs.3,13,14 While hypersensitivity to somatic sensory stimuli represents one aspect of CPSS, it is important to note that CPSS in humans is multifaceted, encompassing various types such as ongoing pain, background pain, spontaneous pain, and pain induced by movement. In particular, LBP and CPSS often impair a patient’s gait and mobility, as patients may compensate for pain or limited range of motion in certain parts.15 Intriguingly, CatWalk gait analysis showed that LBP rats also showed significantly shorter stand time, lower max-intensity, and smaller max-contact area at days 7 and 14 after rod implantation than sham-operated rats. CPSS rats at days 7 and 14 after Step 2 surgery exhibited similar, if not greater, levels of gait impairment. Additionally, in the open field test, the total distance traveled, number of center crossings, and activity distance in the central zone were significantly decreased in LBP rats at day 7 after rod insertion, as compared to sham-operated rats. Similar findings were observed in CPSS rats, suggesting an altered exploratory behavior, a form of active motor activity.

Impaired gait and decreased exploratory behavior could be due to pain, altered locomotor functions, anxiety, and stress, such as that in OA pain and neuropathic pain models.16,17 The LBP rats only showed a trend of impaired passive movement in the rotarod test, indicating preserved muscle strength (Fig. S4). Clinicians recognize that initial symptoms in CPSS patients often present as back or limb pain, while actual muscle strength decline typically occurs in the disease’s mid-to-late stages. Some patients exhibit early signs such as lameness, limited range of motion, and other limb movement constraints resulting from CPSS-induced pain. It remains to be determined which of the aforementioned factors is important to the impaired gait and reduced exploration in LBP and CPSS rats.

An increased excitability of DRG neurons is intricately linked to chronic pain and hyperalgesia.18 Previous studies have highlighted the impact of neuropathic pain on small-diameter DRG neurons, and our electrophysiology recording showed an increased intrinsic excitability of these neurons in CPSS rats, which could make them more responsive to peripheral stimulation, leading to pain hypersensitivity. These alterations can be triggered by inflammatory mediators,19 which lead to the upregulation of N- and T-type calcium channels in DRG neurons.18 In the LBP condition, spine surgery may intensify preexisting inflammatory responses, creating an even more pro-inflammatory environment that impacts adjacent DRGs and induces hyperalgesia and radicular pain. Clinical evidence has revealed the presence of various inflammatory mediators, including IL-6, IL-8, and prostaglandin E2 (PGE2), in wound drainage from patients who have undergone spinal surgery.20,21 In CPSS rats, the laminectomy may result in additional tissue damage and inflammatory responses in close proximity to the lumbar DRGs. Our findings of elevated expression of GFAP in the lumbar DRGs of CPSS rats provide indirect evidence that the pro-inflammatory environment surrounding the surgery site affects neighboring DRGs, which may partly contribute to hyperalgesia and radicular pain. Comparatively, rats in the minimally invasive decompression group (only rod removal) exhibited better behavioral outcomes, suggesting that the hyperalgesia in CPSS rats may be partly due to the extensive tissue damage caused by the laminectomy surgery under LBP condition.

Interestingly, the MOR, which mediates morphine analgesia,22,23 also showed an upregulation in the lumbar DRGs in CPSS rats. Importantly, intraperitoneal administration of DALDA mitigated mechanical and heat hypersensitivity in CPSS rats, with the maximum effect observed 1 h after administration. In line with this finding, patch clamp recording and calcium imaging, which are important and complementary techniques for examining neuronal excitability, showed that DALDA reduced the number and amplitude of action potential firing and suppressed capsaicin-induced increases of [Ca2+]i in L5 DRG neurons from CPSS rats, indicating inhibition of neuron excitability.

In the current study, our primary goal is to test whether DALDA inhibits DRG neuron responses in the CPSS model. Changes in MOR expression in the DRGs of Group 2 and Group 3 rats, which also exhibited varying degrees of pain, warrant further investigation. Nevertheless, we have previously shown that DALDA also inhibited pain behavior and DRG neuronal activity in other pain conditions (e.g., neuropathic pain).22,24 Therefore, it is possible that DALDA may also inhibit neuron responses in other pain groups (e.g., Group 3: Rod retention). Given that the lack of tight junctions in the endothelium of vessels supplying DRG,25,26 increased MOR expression in primary sensory neurons may represent a promising target for enhancing the efficacy of peripherally acting MOR agonists (e.g., DALDA) to inhibit CPSS and avoid severe side effects (e.g., sedation, addiction) associated with the activation MOR in the central nervous system.

In the current study, rats undergoing both rod removal and laminectomy achieved sufficient neural tissue decompression, yet their pain hyperalgesia did not ameliorate during the early post-intervention phase. This observation parallels the clinical scenario where patients, despite undergoing successful decompression procedures for LBP, continue to experience prolonged pain symptoms. This finding provides valuable insights into exploring the mechanisms of non-structural factors contributing to pain persistence. Our new CPSS model may replicate the progression of human LBP from its onset to surgical intervention, culminating in establishing a rat model exhibiting cutaneous hypersensitivity and gait impairment. As such, it may offer a potentially more faithful representation of the natural course of CPSS in humans, compared to previously described models, such as laminectomy alone, hemilaminectomy,5,6 or bilateral dorsal root cutting,7 as these prior models deviate from the clinical setting to varying extents.

CPSS stemming from identifiable structural factors typically necessitates revision surgery.27 Conversely, CPSS arising from non-structural factors lacks clear etiology and effective treatment options. Hence, greater emphasis should be placed on investigating non-structural factors to enhance understanding and therapeutic strategies for this condition. The preparation protocol for this animal model circumvents potential nerve tissue decompression resulting from structural factors by removing the rod and fully alleviating nerve compression via laminectomy. Yet, despite these interventions, the rats still demonstrated hyperalgesia. To rule out the possibility of nerve tissue damage due to laminectomy, we conducted laminectomy procedures in naive rats; however, they did not exhibit pain hyperalgesia. Thus, the primary advantage of this novel model may lie in its heightened clinical resemblance, particularly in mirroring the disease evolution process, and will be suitable for investigating the mechanisms associated with non-structural factors.

Our model still faces challenges in capturing all these nuanced clinical symptoms.28 Although it effectively mirrors hyperalgesia and gait impairment observed in clinical settings, additional behavioral assessment techniques (e.g., spontaneous and ongoing pain) are warranted to comprehensively evaluate other aspects of CPSS. In addition, it’s widely acknowledged that lumbar spine loads are greater in humans compared to quadrupeds, presenting a common challenge for quadrupedal disease models. However, quadruped spines endure continuous anterior-posterior compression from paravertebral muscles and ligaments, thereby sustaining axial stress to some degree. The position of the inserted rod may have slightly changed during free movement. To limit this, we tightly sutured the muscle overlying the rod and observed that the rod remained in the inserted position when we conducted Step 2 surgery in Group 1 and Group 2 rats.

We observed a clear sign of compression in the ipsilateral L5 DRG after rod insertion (Fig. S5). Additionally, by staining for caspase-3, a key enzyme involved in the process of apoptosis or programmed cell death, we observed a significant increase in the percentage of caspase-3 positive neurons in the ipsilateral L5 DRGs of CPSS rats compared to the contralateral side, indicating neuronal damage (Fig. S6). We speculate that this change is more likely caused by chronic compression rather than directly injuring the DRG from the inserted rod, as we standardized and limited the insertion depth of the small blunt rod (tip diameter: 1 mm; length: 6 mm), keeping at least 2–3 mm outside the intervertebral foramen rather than fully inserting it. The etiology of clinical lower back pain is highly complex, and neural tissues are often subjected not only to mechanical compression but also to chemical irritation from intervertebral discs.29 This presents a key challenge for current animal models of lower back pain caused by mechanical nerve compression.

A gender disparity has been noted in CPSS diagnosis, with more women diagnosed compared to men.30 Yet, female and male CPSS rats exhibit similar mechanical and heat hypersensitivity. This discrepancy may be partly due to different outcome measures and species differences that may influence the progression of CPSS.31,32 For example, clinicians typically diagnose CPSS if pain persists for 6–12 months following spinal surgery.33 Yet, CPSS rats displayed the most significant pain hypersensitivity in the initial 2–3 weeks after rod removal and laminectomy. Although a rat’s 30-day life is equivalent to one human year,31,32 the relatively quick onset and limited duration of evoked pain hypersensitivity in CPSS rats suggest this model may partially mirror the progression of CPSS in humans. In addition, CPSS in patients involves multifactorial considerations, including emotional and social factors,34 posing challenges in fully replicating and analyzing these complexities in animal models.

Rats possess remarkable self-healing abilities.35,36 Their peripheral nervous system also shows greater resistance to injury, enabling faster recovery from nerve damage.35 Unlike humans, where nerve compression often exacerbates symptoms with activity, rats typically experience a quicker reduction in hypersensitivity following similar injuries, such as in DRG compression models.36 The differences in recovery between rats and humans are thought to stem from species-specific variations in healing processes, including enhanced angiogenesis, increased cell proliferation, and reduced inflammatory responses.35,36 Therefore, species-specific differences should be taken into consideration when assessing treatment outcomes in rats, especially when simulating clinical treatment protocols.

Among the various pain-related genes in the DRG, we assessed P2X7 and GFAP, which are mainly expressed in non-neuronal cells (e.g., satellite glial cells) in the ganglion, TRPA1, which is an important receptor extensively expressed in nociceptive DRG neurons and MOR which is a potential target on DRG neurons for inhibiting CPSS with peripherally acting MOR agonists. This represents an initial exploration into characterizing the molecular basis of this new CPSS model. There are many other receptors on DRG neurons that are important for pain. Understanding their transcriptional and translational roles in CPSS is essential for developing targeted therapies. Full-scale studies on the cellular and molecular mechanisms of CPSS are warranted, including comprehensive profiling of changes in gene and protein expression of other receptors in DRG neurons and glial cells, investigating the signaling pathways, and examining the effects of other analgesics used in clinical pain management (e.g., gabapentin, clonidine) to provide deeper insights into the pathophysiology of CPSS.