An Automated Cell Detection Method for TH-positive Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease Using Convolutional Neural Networks

Quantification of tyrosine hydroxylase (TH)-positive neurons is essential for the preclinical study of Parkinson’s disease (PD). However, manual analysis of immunohistochemical (IHC) images is labor-intensive and has less reproducibility due to the lack of objectivity. Therefore, several automated methods of IHC image analysis have been proposed, although they have limitations of low accuracy and difficulties in practical use. Here, we developed a convolutional neural network-based machine learning algorithm for TH+ cell counting. The developed analytical tool showed higher accuracy than the conventional methods and could be used under diverse experimental conditions of image staining intensity, brightness, and contrast. Our automated cell detection algorithm is available for free and has an intelligible graphical user interface for cell counting to assist practical applications. Overall, we expect that the proposed TH+ cell counting tool will promote preclinical PD research by saving time and enabling objective analysis of IHC images.

Adenovirus-mediated Foxp3 expression in lung epithelial cells reduces airway inflammation in ovalbumin and cockroach-induced asthma model

Foxp3 is a master regulator of CD4⁺CD25⁺ regulatory T-cell (Treg) function and is also a suppressor of SKP2 and HER2/ErbB2. There are an increasing number of reports describing the functions of Foxp3 in cell types other than Tregs. In this context, we evaluated the functions of Foxp3 in ovalbumin- and cockroach-induced asthma models. Foxp3-EGFP-expressing adenovirus or EGFP control adenovirus was administered intratracheally (i.t.), followed by challenge with ovalbumin (OVA) or cockroach extract to induce asthma. Th2 cytokine and immune cell profiles of bronchoalveolar lavage fluid (BALF), as well as serum IgE levels, were analyzed. Histological analyses were also conducted to demonstrate the effects of Foxp3 expression on airway remodeling, goblet cell hyperplasia and inflammatory responses in the lung. Adenoviral Foxp3 was expressed only in lung epithelial cells, and not in CD4⁺ or CD8⁺ cells. BALF from Foxp3 gene-delivered mice showed significantly reduced numbers of total immune cells, eosinophils, neutrophils, macrophages and lymphocytes in response to cockroach allergen or OVA. In addition, Foxp3 expression in the lung reduced the levels of Th2 cytokines and IgE in BALF and serum, respectively. Moreover, histopathological analysis also showed that Foxp3 expression substantially inhibited eosinophil infiltration into the airways, goblet cell hyperplasia and smooth muscle cell hypertrophy. Furthermore, when Tregs were depleted by diphtheria toxin in Foxp3DTR mice, the anti-asthmatic functions of Foxp3 were not altered in OVA-challenged asthma models. In this study, our results suggest that Foxp3 expression in lung epithelial cells, and not in Tregs, inhibited OVA- and cockroach extract-induced asthma.

Bee venom phospholipase A2 ameliorates motor dysfunction and modulates microglia activation in Parkinson's disease alpha-synuclein transgenic mice

α-Synuclein (α-Syn) has a critical role in microglia-mediated neuroinflammation, which leads to the development of Parkinson's disease (PD). Recent studies have shown that bee venom (BV) has beneficial effects on PD symptoms in human patients or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin-induced PD mice. This study investigated whether treatment with BV-derived phospholipase A2 (bvPLA2) would improve the motor dysfunction and pathological features of PD in human A53T α-Syn mutant transgenic (A53T Tg) mice. The motor dysfunction of A53T Tg mice was assessed using the pole test. The levels of α-Syn, microglia and the M1/M2 phenotype in the spinal cord were evaluated by immunofluorescence. bvPLA2 treatment significantly ameliorated motor dysfunction in A53T Tg mice. In addition, bvPLA2 significantly reduced the expression of α-Syn, the activation and numbers of microglia, and the ratio of M1/M2 in A53T Tg mice. These results suggest that bvPLA2 could be a promising treatment option for PD.

Immunotherapy with methyl gallate, an inhibitor of Treg cell migration, enhances the anti-cancer effect of cisplatin therapy

Foxp3+ CD25+CD4+ regulatory T (Treg) cells are crucial for the maintenance of immunological self-tolerance and are abundant in tumors. Most of these cells are chemo-attracted to tumor tissues and suppress anti-tumor responses inside the tumor. Currently, several cancer immunotherapies targeting Treg cells are being clinically tested. Cisplatin is one of the most potent chemotherapy drugs widely used for cancer treatment. While cisplatin is a powerful drug for the treatment of multiple cancers, there are obstacles that limit its use, such as renal dysfunction and the development of cisplatin-resistant cancer cells after its use. To minimize these barriers, combinatorial therapies of cisplatin with other drugs have been developed and have proven to be more effective to treat cancer. In the present study, we evaluated the eff ect of the combination therapy using methyl gallate with cisplatin in EL4 murine lymphoma bearing C57BL/6 mice. The combinatorial therapy of methyl gallate and cisplatin showed stronger anti-cancer eff ects than methyl gallate or cisplatin as single treatments. In Treg cell-depleted mice, however, the eff ect of methyl gallate vanished. It was found that methyl gallate treatment inhibited Treg cell migration into the tumor regardless of cisplatin treatment. Additionally, in both the normal and cisplatin-treated tumor-bearing mice, there was no renal toxicity attributed to methyl gallate treatment. These findings suggest that methyl gallate treatment could be useful as an adjuvant method accompanied with cisplatin therapy.

Neuroprotective Effects of AMP-Activated Protein Kinase on Scopolamine Induced Memory Impairment

AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, is activated in response to cellular stress when intracellular levels of AMP increase. We investigated the neuroprotective effects of AMPK against scopolamine-induced memory impairment in vivo and glutamate-induced cytotoxicity in vitro. An adenovirus expressing AMPK wild type alpha subunit (WT) or a dominant negative form (DN) was injected into the hippocampus of rats using a stereotaxic apparatus. The AMPK WT-injected rats showed significant reversal of the scopolamine induced cognitive deficit as evaluated by escape latency in the Morris water maze. In addition, they showed enhanced acetylcholinesterase (AChE)-reactive neurons in the hippocampus, implying increased cholinergic activity in response to AMPK. We also studied the cellular mechanism by which AMPK protects against glutamate-induced cell death in primary cultured rat hippocampal neurons. We further demonstrated that AMPK WT-infected cells increased cell viability and reduced Annexin V positive hippocampal neurons. Western blot analysis indicated that AMPK WT-infected cells reduced the expression of Bax and had no effects on Bcl-2, which resulted in a decreased Bax/Bcl-2 ratio. These data suggest that AMPK is a useful cognitive impairment treatment target, and that its beneficial effects are mediated via the protective capacity of hippocampal neurons.

Electroacupuncture Analgesia Is Improved by Adenoviral Gene Transfer of Dopamine Beta-hydroxylase into the Hypothalamus of Rats

Electroacupuncture (EA) is a modified form of acupuncture that utilizes electrical stimulation. We previously showed that EA stimulated rats were divided into responders that were sensitive to EA and non-responders that were insensitive to EA based on the tail flick latency (TFL) test. The dopamine beta-hydroxylase (DBH) gene was more abundantly expressed in the hypothalamus of responder rats than non-responder rats. To determine whether overexpression of DBH gene expression in the hypothalamus modulate EA analgesia, we constructed a DBH encoding adenovirus and which was then injected into the hypothalamus of SD rats. Microinjection of DBH or control GFP virus into the hypothalamus had no changes on the basal pain threshold measured by a TFL test without EA treatment. However, the analgesic effect of EA was significantly enhanced from seven days after microinjection of the DBH virus, but not after injection of the control GFP virus. DBH expression was significantly higher in the hypothalamus of DBH virus injected rat than control GFP virus or PBS injected rats. Moreover, expression of the DBH gene did not affect the body core temperature, body weight, motor function or learning and memory ability. Although the functional role of DBH in the hypothalamus in the analgesic effect of EA remains unclear, our findings suggest that expression of the DBH gene in the hypothalamus promotes EA analgesia without obvious side-effects.

Resveratrol stimulates glucose transport in C2C12 myotubes by activating AMP-activated protein kinase

trans-Resveratrol (t-RVT), a naturally occurring polyphenol found in Polygonum cuspidatum, grape, and red wine, has been reported to have anti- inflammatory, cardioprotective, and cancer chemopreventive properties. However antidiabetic effect of t-RVT has not yet been reported. In this study, we show that t-RVT increases glucose uptake in C2C12 myotubes by activating AMP-activated protein kinase (AMPK), uncovering an antidiabetic potential of t-RVT for the first time. AMPK plays a central role in the regulation of glucose and lipid metabolism, and hence it is considered a novel therapeutic target for metabolic syndrome such as type 2 diabetes. t-RVT significantly induced glucose uptake in C2C12 cells, via AMPK activation, but not a phosphatidylinositol-3 kinase (PI-3 kinase) signal pathway. The induced glucose uptake was attenuated by pretreatment with a pharmacological inhibitor for AMPK, indicating that the effect of t-RVT primarily depends on AMPK activation. However, in the presence of insulin, t-RVT also potentiated the effect of insulin on glucose uptake via AMPK activation, which led to further activation of PI-3 kinase/Akt signal pathway.