The Analgesic Studies Of Selective Blocking Of Descending Facilitation Through μ Opioid Receptor Mediated Targeted Lesion In The Rat Model Of Cancer Induced Bone Pain

BackgroundAs advances in cancer detection and therapy extend the life expectancy of cancer patients, there is an increasing focus on improving patients'quality of life. Bone cancer pain is the most common pain in patients with advanced cancer as most common tumors including breast, prostate, and lung cancer have a remarkable affinity to metastasize to bone. Once tumors metastasize to bone, they are usually major causes of morbidity or mortality, and consequently compromise patients'survival and quality of life. Currently, factors that drive cancer pain are poorly understood; however, in the just past decade several animal models of cancer induced bone pain (CIBP) have been developed. In terms of tumor growth, bone remodeling, and bone pain, these models seem to mirror several aspects of human bone caner pain, providing insight into mechanisms that drive bone cancer pain. The researchers realize by degree that bone cancer pain is a unique persistent pain state, including elements of both neuropathy and inflammation.Currently, the treatment of pain from bone metastases, involving the use of multiple complementary approaches, is frequently relatively ineffective. And the neurobiological basis for the treatment is largely empirical or based on scientific studies arising from some aspects of cancer pain. In clinic, the severity of this pain is variable from patient to patient, tumor to tumor, and time to time, even from site to site plausibly because their underling mechanisms are inconsistent. In addition, the metastases are generally not limited to a single site and analgesics that are most commonly used to treat bone caner pain are limited by significant adverse side effects. Therefore, it is a big challenge for us to explore some novel mechanism-based approaches to relieve bone cancer pain for most patients.Complex networks of pathways project from various supraspinal structures to modulate spinal processing of sensory input in a top-down fashion. The rostral ventromedial medulla (RVM) in the brainstem is one major final common output of this endogenous modulatory system and is involved in the relay of sensory information between the spinal cord and brain. The net output of descending neurons that exert inhibitory or facilitatory effects will determine whether neuronal activity in the spinal cord increased or decreased. Extensive behavioral and electrophysiological studies suggest that descending facilitation from the RVM is critical for maintenance of central sensitization in neuropathic pain. Thus, targeting these descending facilitatory neurons may be a novel and as yet clinically unexploited method for treating chronic intractable pain. However, little is known that whether this modality is also workable in CIBP. Thus, through selective ablating ablationμreceptor positive facilitatory neurons with dermorphin-saporin, this research investigated analgesic effects through blocking descending facilitation from RVM in the rat model of CIBP and evaluated the safety of this targeted lesion. Furthermore, considering complex making and high price of dermorphin-saporin, we continued to clone a rat constitutively active recombinant caspase-3 gene in order to lay the foundation for future constructing dermorphin and constitutive active recombinant caspase-3 gene conjugate for gene therapy of CIBP. Methods and Results1. Inhibition of cancer induced bone pain through selective ablation ofμ-opioid receptor mediated descending facilitation from the rostral ventromedial medulla Methods: A total of 96 adult female Wistar rats were randomly divided into six groups: 1) na?ve group (n=6, without any intervention); 2) carcinoma cell group (n=18, without any intra-RVM microinjection); 3) PBS group (n=18, received a single intra-RVM microinjection with PBS alone); 4) dermorphin group (n=18, received a single intra-RVM microinjection with dermorphin); 5)saporin group (n=18, received a single intra-RVM microinjection with saporin); 6) dermorphin-saporin group (n=18, received a single intra-RVM microinjection with dermorphin-saporin). On day 28 post-microinjection, all rats except na?ve rats were inoculated with Walker 256 carcinoma cells into the right tibiae. Commencing from day 3 to day 20 post-inoculation, mechanical allodynia, mechanical hyperalgesia, cold allodynia, thermal hyperalgesia and the score of ambulatory pain were recorded to investigate changes of nociceptive behaviors. The development of the bone tumor and structural destruction to the bone was monitored by radiological analysis. After repetitive non-noxious tactile stimulation, the total number of FOS positive neurons in the spinal cord horn was regarded as a marker indicative of central sensitization. The GFAP (Glial fibrillary acidicprotein) expression in the spinal cord was detected through immunohistochemistry. And changes of proinflammatory cytokins, such as IL-1βand TNF-αwere observed by ELISA.Results: 1) Nociceptive behaviors: Rats in carcinoma cells group, PBS group, dermorphin group or saporin group demonstrated nociceptive hypersensitivity compared with na?ve rats (P<0.05), while rats in dermorphin-saporin group showed decreased behaviors within 4-7 days after the onset of nociception in comparison with carcinoma cells group (P<0.05). 2) Radiological analysis: All of rats inoculated with carcinoma cells showed ongoing damage to cortical bone and the trabeculae in the ipsilateral tibial bone from day 7 post-inoculation, and by day 20 the damage threatened the integrity of the tibial bone. All intra-RVM treatments failed to influence the bone destruction during the observation. 3) FOS positive neurons: In parallel with behavioral tests, the number of FOS positive neurons in the bilateral spinal cord significantly enhanced in all groups treated with carcinoma cells compared with na?ve rats on day 7 post-inoculation (P<0.05), whereas dermorphin-saporin declined significantly the amount of FOS labeled neurons on day 14 and day 20 post-inoculation compared with non-microinjection or other intra-RVM treatment (P<0.05). 4) The amount of activated spinal astrocytes and the protein levels of proinflammatory cytokines: The amount of activated astrocytes and the protein levels of IL-1βor TNF-αin the bilateral spinal cord from carcinoma cell group, PBS group, dermorphin group and saporin group increased significantly compared with that from na?ve rats (P<0.05). In contrast, intra-RVM microinjection with dermorphin-saporin evidently decreased the amount of activated astrocytes and down-regulated the production of proinflammatory cytokines relative to non-microinjection (P<0.05), and reversed them to the baseline levels on day 20 post-inoculation (P>0.05).2. Safety evaluation of microinjection of the targeted neuropepetide-toxin conjugate, dermophine-saporin, into the rat rostral ventromedial medullaMethods :A total of 102 adult Wistar rats were randomly divided into five groups: 1) na?ve group(n=6, without any intervention); 2) PBS group (n=24, received a single intra-RVM microinjection with PBS alone); 3) dermorphin group (n=24, received a single intra-RVM microinjection with dermorphin); 4)saporin group (n=24, received a single intra-RVM microinjection with saporin); 5) dermorphin-saporin group (n=24, received a single intra-RVM microinjection with dermorphin-saporin). From day 4 to day 28 after microinjection, physiological parameters, such as body weight, rectal temperature, respiratory rate, heart rate and systolic blood pressure, were observed every 4 days. On day 4, 7, 14 and 28 post-microinjection, cardiovascular functions were recorded through echocardiogram. Meanwhile, both the number of neurons and the activity of glia were determined via immunohistochemistry, and the mRNA and protein levels of proinflammatory cytokines, such as IL-1βand TNF-α, were analyzed by real-time PCR and ELISA, respectively.Results: 1) Intra-RVM with dermorphin-saporin had no significant influence on the physiological parameters (P>0.05), including body weight, rectal temperature, respiratory rate, heart rate and systolic blood pressure; 2) With echocardiogram there was no significant difference in EF (left ejection fraction), FS (left fractional shortening), LVIDS (left ventricular internal dimension systole at end-diastole), and LVIDD (left ventricular internal dimension diastole at end-diastole) between dermorphin-saporin group and na?ve group on day 7, day 14 and day 28 post-microinjection (P>0.05). However, EF and FS significantly increased in all of groups receiving intra-RVM microinjection compared with na?ve group on day 4 after microinjection (P<0.05); 3) The total number of NeuN labeled neurons in the RVM failed to exert significant differences between dermorphin-saporin group and niave group (P>0.05). 4) In the local micro-environment the resident microglia or astrocytes kept quiescent, and the mRNA or protein levels of IL-1βor TNF-αmaintained at the baseline during the observation (P>0.05).3. Construction of rat constitutively active recombinant caspase-3 and evaluation of its apoptotic effects in vitroMethods: The rat constitutively active recombinant caspase-3 gene was constructed through reversing two subunits of rat wild type caspase-3 gene by overlap-PCR, and cloned into expression vector EGFP-C1 or pcDNA3.1(+).Then, the recombinant caspase-3 was transfected into human 293T cells or rat immortalized neural progenitor cells. The target gene expression and the morphology of transfected cells were observed by fluorescence microscope, and apoptotic effects of the recombinant gene were analyzed by Annexin V-Fluorescein∕PI flow cytometry, MTT assay and Western blot. Results: The recombinant caspase-3 can be expressed efficiently in both 293T cells and INPC. The transfected cells with recombinant caspse-3 gene presented typical characteristics of apoptosis, including cellular fragmentation and cytoplasm concentration with fluorescence microscope. Results from Annexin V-Fluorescein∕PI flow cytometry suggested the gene resulted significantly in early apoptosis in both cells compared with controls (P<0.05). In MTT assay both cells displayed evident growth inhibition after transfection with the recombinant caspase-3 relative to controls (P<0.05). And the protein levels of active caspase-3 increased significantly in cells transfected with the recombinant capase-3 compared to cells with other treatment (P<0.05).4. Statistical analysisAll of analyses were performed by SigmaStat 3.0 software package. All data were expressed as the mean±standard deviation (SD). Significant differences within the first part were detected by two-factor ANOVA followed by post-hoc test (SNK), and one-way ANOVA was utilized to detect significant differcence in other parts followed by SNK. Significance was set at P<0.05.Conclusion1. In our rat model of CIBP, selective ablation ofμopioid receptor expressing descending facilitatory neurons in the RVM with dermorphin-saporin reduced a variety of nociceptive behaviors through inhibiting spinal sensitization, decreasing the amount of activated astrocytes and down-regulating the proinflammatory cytokines (IL-1βor TNF-α) production.2. Microinjection of dermorphin-saporin into the RVM had no significant impacts on the physiological functions and heart functions. And neither significant loss of NeuN labelsled neurons nor evident responses of the neuroinflammtory or neuroimmune were absent during the observation.3. The constitutively active rat recombinant caspase-3 induced apoptosis without pro-apoptotic signals stimulation, and imitated cell killing effects of saporin in the targeted cells.SignificanceAccumulating evidence suggests that inappropriate tonic-descending facilitation arising from the RVM in the brainstem has been established to underlie some chronic pathologic pain. Our research selectively ablatedμopioid receptor expressing neurons in the RVM, presumed to be a source of spinopetal facilitatory projection, by a single intra-RVM microinjection with dermorphin-saporin, and subsequently reversed the experimental CIBP effectively. On the other hand, results of this research established the targeted lesion is a safe and reliable approach to treat pathologic pain. Thus, this research is a novel exploration of mechanism-based therapy for CIBP, providing us some experimental evidence for future clinical application. Furthermore, considering the drawbacks of saporin, we mimicked cell killing effects of saporin in the targeted cells, and cloned the constitutively active recombinant caspase-3, laying the foundation for our future targeted gene therapies in CIBP.