An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND: The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS: The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION: Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles.

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis.

Protective effects of dihydromyricetin on primary hippocampal astrocytes from cytotoxicity induced by comorbid diabetic neuropathic pain and depression.

Activated astrocytes play a key role in diabetic neuropathic pain and depression. We aimed to assess the protective effects of dihydromyricetin (DHM) on primary hippocampal astrocytes cultured with high glucose (HG), substance P (SP), and corticosterone (CORT). Culturing with HG + SP + CORT resulted in damage to primary hippocampal astrocytes, which simulates the clinical damage caused by comorbidity of diabetic neuropathic pain and depression. Western blot, qPCR, and immunofluorescence analyses revealed that HG + SP + CORT increased P2X7 receptor expression in primary hippocampal astrocytes, which was reversed by DHM treatment. Further, HG + SP + CORT elevated TNF-α, IL-1ß, free Ca2+, and ERK1/2 phosphorylation levels, which was inhibited by DHM or P2X7 shRNA treatment. Moreover, DHM significantly reduced the P2X7 agonist-activated currents in HEK293 cells transfected with the P2X7 receptor. These findings suggest that DHM can protect primary hippocampal astrocytes cultured with HG + SP + CORT from P2X7 receptor-mediated damage. Culturing cells with HG + SP + CORT might be a viable cell model for cellular injury exploration of diabetic comorbid pain and depression.

Effects of palmatine on BDNF/TrkB-mediated trigeminal neuralgia.

Trigeminal neuralgia (TN), a sudden, needle-like pain in the distribution area of the trigeminal nerve, can seriously affect the physical and mental health of patients. In chronic pain conditions including TN, increased levels of brain-derived neurotrophic factor (BDNF) may enhance pain transmission. This study compares the effect of palmatine administration on the expression of BDNF and its receptor TrkB (tropomyosin receptor kinase B) in trigeminal ganglion cells of Sprague-Dawley rats in a sham versus TN model group. Within 14 days of surgery, the mechanical allodynia threshold of the TN group was significantly lower than that of the sham group, while the TN + palmatine group had a higher mechanical pain sensitivity threshold than the TN group (p < 0.05). Real-time quantitative PCR, immunohistochemistry, and immunofluorescence showed that BDNF and TrkB expression in the TN group was higher than that in the sham group, while palmatine treatment could reverse these changes. Western blotting showed that palmatine treatment could reduce the elevated phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) in TN rats. Thus, the BDNF/TrkB pathway may be involved in the pain transmission process of TN, and palmatine treatment may reduce pain transmission by inhibiting the BDNF/TrkB pathway and suppressing ERK1/2 phosphorylation.

Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X7 Receptor.

Diabetic neuropathic pain (DNP) and major depressive disorder (MDD) are common complications of diabetes mellitus and mutually affect each other. As a member of the ATP-gated ion channel family, P2X7 receptor is associated with the transduction of pain signal and the onset of depression. The aim of this study was to investigate the effects of dihydromyricetin (DHM) on rats with comorbid DNP and MDD. After the comorbid model was established, rat behavior changes were monitored by measuring the mechanical withdrawal threshold, thermal withdrawal latency, sugar water preference, immobility time in the forced-swim test, and open-field test parameters. The expressions of P2X7 receptor in the dorsal root ganglia (DRGs), spinal cord, and hippocampus were assessed by quantitative real-time PCR, Western blotting, and double immunofluorescence. We found that hyperalgesia, allodynia, and depressive behaviors of rats with comorbid DNP and MDD were relieved by treatment with DHM or application of a short-hairpin RNA for P2X7 receptor. The expression levels of P2X7, phosphorylated extracellular signal-regulated kinase 1/2, tumor necrosis factor α, and interleukin 1ß were increased in the DRGs, spinal cord, and hippocampus of rats in the model group but restored after DHM or P2X7 short-hairpin RNA treatment. In conclusion, P2X7 receptor in the DRGs, spinal cord, and hippocampus participates in the transduction of DNP and MDD signals. DHM seems to relieve comorbid DNP and MDD by reducing the expression of P2X7 receptor in the DRGs, spinal cord, and hippocampus and may be an effective new drug for the treatment of patients with both DNP and MDD.

Dihydromyricetin affects BDNF levels in the nervous system in rats with comorbid diabetic neuropathic pain and depression.

Diabetic neuropathic pain (DNP) and depression (DP) are the common complications in patients with diabetes. The purpose of our research was to observe whether brain-derived neurotrophic factor (BDNF) levels and tropomyosin receptor kinase B (TrkB) in the nervous system have effects on rats with comorbid DNP and DP, and to determine whether dihydromyricetin (DHM) may influence BDNF/ TrkB pathway to mitigatethe comorbidity. The study showed that DHM treatment could attenuates pain and depressive behavior in DNP and DP combined rats. Compared with the control group, the expression level of BDNF/TrkB in the hippocampus of DNP + DP group were reduced, while the expression levels in the spinal cord and DRG were increased. However, after treatment with DHM, those changes were reversed. Compared with the control group, the level of IL-1ß and TNF-α in the hippocampus, spinal cord and DRG in the DNP + DP group was significantly increased, and DHM treatment could reduce the increase. Thus our study indicated that DHM can relief symptoms of DNP and DP by suppressing the BDNF/TrkB pathway and the proinflammatory factor, and BDNF/TrkB pathway may be an effective target for treatment of comorbid DNP and DP.

Selecting an Appropriate Animal Model of Depression.

Depression has become one of the most severe psychiatric disorders and endangers the health of living beings all over the world. In order to explore the molecular mechanism that underlies depression, different kinds of animal models of depression are used in laboratory experiments. However, a credible and reasonable animal model that is capable of imitating the pathologic mechanism of depression in mankind has yet to be found, resulting in a barrier to further investigation of depression. Nevertheless, it is possible to explain the pathologic mechanism of depression to a great extent by a rational modeling method and behavioral testing. This review aims to provide a reference for researchers by comparing the advantages and disadvantages of some common animal depression models.

Effects of long non-coding RNA uc.48+ on pain transmission in trigeminal neuralgia.

Trigeminal neuralgia (TN) is the most common neuropathic pain in the facial area, for which the effective therapy is unavailable. Long non-coding RNA (lncRNA) such as lncRNA uc.48+ is involved in diabetic neuropathic pain and may affect purinergic signaling in ganglia of diabetic rats. In this research, chronic constriction injury of the infraorbital nerve (CCI-ION) was applied to establish a rat model of TN. Five days after local injection of siRNA targeting the lncRNA uc.48+ in trigeminal ganglia (TGs), the upregulated uc.48+ expression and the reduced mechanical withdrawal threshold (MWT) in the TN rats were significantly reversed. The expression of P2X7 receptor in TGs was increased in the TN group compared with the sham group, but uc.48+ siRNA treatment mitigated this effect. The phosphorylation of ERK1/2 in TGs of TN rats was significantly enhanced compared with the sham group, while uc.48+ siRNA treatment reversed this change. In addition, injection of the lncRNA uc.48+ overexpression plasmid in TGs of control rats significantly reduced the MWT but elevated the expression of P2X7 in TGs; the phosphorylation of ERK1/2 in TGs in these uc.48+-overexpressed rats was significantly higher, similar to the observations in rats of TN model. The interaction between uc.48+ and the P2X7 receptor was detected by RNA binding protein immunoprecipitation (RIP), indicating that P2X7 receptor could specifically bind to uc.48 + . In summary, knockdown of lncRNA uc.48+ by siRNA could inhibit transduction of TN signals, whereas uc.48+ overexpression promoted TN signal transduction. LncRNA uc.48+ may interact with P2X7 receptor to upregulate expression of P2X7 receptor and furthermore enhance the phosphorylation of ERK1/2 in TGs, thereby participating in pain transmission in TN.

Effects of palmatine on rats with comorbidity of diabetic neuropathic pain and depression.

Diabetic neuropathic pain (DNP) is one of the common complications of diabetes. Depression (DP) is also one of the common complications of diabetes. P2X7 receptors play an important role in the transmission of nociceptive signal and are associated with depressive illness. In the study, the hyperalgesia, allodynia and depressive behaviours of rats with comorbidity of DNP and DP were confirmed by the thermal withdrawal latency (TWL) test, mechanical withdrawal threshold (MWT) test, sucrose preference test (SPT), immobility time of forced swimming test (IMFST) and open-field test (OFT). The change in expression of the P2X7 receptor of the hippocampus was observed through RT-PCR, qPCR, Western blotting and immunohistochemical staining methods The results showed that palmatine treatment can alleviate the hyperalgesia, allodynia and depressive behaviours of rats with comorbidity of DNP and DP. Meanwhile, the expression of P2X7 receptors, GFAP, TNF-α and IL-1ß in the hippocampus of the rats with comorbidity of DNP and DP was significantly increased compared with the control rats, and palmatine treatment could decrease the expression. Furthermore, the enhanced phosphorylation of ERK1/2 in the hippocampus of rats with DNP and DP was decreased noticeably by palmatine treatment. The results of this study suggest that palmatine can alleviate the comorbidity of DNP and DP by inhibiting the expression of P2X7 receptors in the hippocampus, and its action may be related to suppression of the phosphorylation of ERK1/2 and the release of TNF-α and IL-1ß in the hippocampus.

Emodin inhibits the expression of receptor and calcitonin-gene-related peptide release in trigeminal ganglia of trigeminal neuralgia rats.

Trigeminal neuralgia (TN) is one of the most intense forms of facial pain. It has been reported that the P2X3 receptor plays a crucial role in facilitating pain transmission, and the calcitonin-gene-related peptide (CGRP) from trigeminal ganglia (TGs) might perform differing function in nociceptive afferent input transmission. The present study investigated whether emodin can affect TN pain transmission by suppressing the expression of P2X3 receptors and CGRP in TGs. Chronic constriction injury of the infraorbital branch of the trigeminal nerve (CCI-ION) was used as TN model. The TN rats were randomly divided into the following 4 groups: (1) a sham group (Sham), (2) a sham rats treated with emodin group (TN + E), (3) a TN rats treated with 0.5% sodium carboxymethyl cellulose (CMC) as vehicle group (TN) and (4) a TN rats treated with emodin group (TN + E). The mechanical hyperalgesia threshold of TN rats was tested by Electric Von Frey filaments. The change of the expression of P2X3 receptors and CGRP in rat's TG was detected with RT-PCR, immunohistochemical staining, and Western blotting. The phosphorylation of p38 and ERK1/2 pathway of TG was detected by Western blotting. After CCI-ION injury, the threshold of mechanical hyperalgesia for the territory of ligated infraorbital nerve in TN group decreased significantly compared with that in sham group. On day 14 after operation of CCI-ION, there was also an evident increase in the expression of P2X3 receptors and CGRP in the TG of TN group. However after treatment with emodin, the response of mechanical hyperalgesia of TN rats was clearly increased while the enhanced expression of P2X3 receptor and CGRP in TN rats was significantly decreased. The phosphorylation of p38 and ERK1/2 in TN group was stronger than that in Sham group. But these phosphorylation changes in the TN rats were much weaker after treatment with emodin. In conclusion, P2X3 receptor may cooperate with CGRP in the pain transmission of TN, and emodin can inhibit the expression and activation of P2X3 receptor and CGRP in TG to relieve TN.