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.

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.

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.