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Browsing by Author "Bhatwadekar, Ashay"
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Item Computer-based Quantification of Acellular Capillaries to Assess Experimental Diabetic Retinopathy(Office of the Vice Chancellor for Research, 2016-04-08) Hemmady, Anish; Tuceryan, Mihran; Bhatwadekar, AshayDiabetic retinopathy (DR) is a disease of small blood vessels in the retina. The increase in the number of acellular capillaries is used as a marker to assess the severity of DR. The traditional approach for identifying acellular capillaries is manual counting of the capillaries either directly under the microscope or using the captured images. However, these methods are cumbersome and often involve inconsistencies among researchers. The purpose of this study is to reduce discrepancies in the enumeration of acellular capillaries using computer-based image processing algorithms. The retinas of control and diabetic mice were processed using trypsin digestion. The high resolution png format images of retinal quadrants were prepared from the trypsin digested retina. The computer programming was performed using the Python language along with open source packages such as OpenCv, Python Imaging Library (PIL), NumPy (Numerical Python) and SciPy. The images initially corrected for a Gaussian Blur and a Median blur to remove noise followed by the histogram based image segmentation. After image segmentation, a binary image was generated based on a histogram analysis. The segmentation threshold for binary image was determined and the medial axis transform (MAT) algorithm was applied to the binary image. The MAT representation was used to skeletonize the blood vessels and to detect branches and branch-points in those blood vessels. As part of the MAT computation, the distances from the skeleton to the vessel boundaries are encoded. The thin capilleries, i.e., acellular capilleries, were identified using a threshold on this distance which encodes the thickness of the vessel. Finally, acellular capillaries were counted by connected component algorithm. In conclusion, we have designed an automated computer-based system to enumerate the acellular capillaries. This computer-based automated system will help to maintain consistency in retinopathy assessment and may reduce time for analysis.Item Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells(Wiley, 2017-05) Salazar, Tatiana E.; Richardson, Matthew R.; Beli, Eleni; Ripsch, Matthew S.; George, John; Kim, Youngsook; Duan, Yaqian; Moldovan, Leni; Yan, Yuanqing; Bhatwadekar, Ashay; Jadhav, Vaishnavi; Smith, Jared A.; McGorray, Susan; Bertone, Alicia L.; Traktuev, Dmitri O.; March, Keith L.; Colon-Perez, Luis M.; Avin, Keith; Sims, Emily; Mund, Julie A.; Case, Jamie; Deng, Shaolin; Kim, Min Su; McDavitt, Bruce; Boulton, Michael E.; Thinschmidt, Jeffrey; Calzi, Sergio Li; Fitz, Stephanie D.; Fuchs, Robyn K.; Warden, Stuart J.; McKinley, Todd; Shekhar, Anantha; Febo, Marcelo; Johnson, Phillip L.; Chang, Lung Ji; Gao, Zhanguo; Kolonin, Mikhail G.; Lai, Song; Ma, Jinfeng; Dong, Xinzhong; White, Fletcher A.; Xie, Huisheng; Yoder, Mervin C.; Grant, Maria B.; Ophthalmology, School of MedicineElectroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief.Item Restructuring of the Gut Microbiome by Intermittent Fasting Prevents Retinopathy and Prolongs Survival in db/db Mice(American Diabetes Association, 2018-09) Beli, Eleni; Yan, Yuanqing; Moldovan, Leni; Vieira, Cristiano P.; Gao, Ruli; Duan, Yaqian; Prasad, Ram; Bhatwadekar, Ashay; White, Fletcher A.; Townsend, Steven D.; Chan, Luisa; Ryan, Caitlin N.; Morton, Daniel; Moldovan, Emil G.; Chu, Fang-I; Oudit, Gavin Y.; Derendorf, Hartmut; Adorini, Luciano; Wang, Xiaoxin X.; Evans-Molina, Carmella; Mirmira, Raghavendra G.; Boulton, Michael E.; Yoder, Mervin C.; Li, Qiuhong; Levi, Moshe; Busik, Julia V.; Grant, Maria B.; Pediatrics, School of MedicineIntermittent fasting (IF) protects against the development of metabolic diseases and cancer, but whether it can prevent diabetic microvascular complications is not known. In db/db mice, we examined the impact of long-term IF on diabetic retinopathy (DR). Despite no change in glycated hemoglobin, db/db mice on the IF regimen displayed significantly longer survival and a reduction in DR end points, including acellular capillaries and leukocyte infiltration. We hypothesized that IF-mediated changes in the gut microbiota would produce beneficial metabolites and prevent the development of DR. Microbiome analysis revealed increased levels of Firmicutes and decreased Bacteroidetes and Verrucomicrobia. Compared with db/db mice on ad libitum feeding, changes in the microbiome of the db/db mice on IF were associated with increases in gut mucin, goblet cell number, villi length, and reductions in plasma peptidoglycan. Consistent with the known modulatory effects of Firmicutes on bile acid (BA) metabolism, measurement of BAs demonstrated a significant increase of tauroursodeoxycholate (TUDCA), a neuroprotective BA, in db/db on IF but not in db/db on AL feeding. TGR5, the TUDCA receptor, was found in the retinal primary ganglion cells. Expression of TGR5 did not change with IF or diabetes. However, IF reduced retinal TNF-α mRNA, which is a downstream target of TGR5 activation. Pharmacological activation of TGR5 using INT-767 prevented DR in a second diabetic mouse model. These findings support the concept that IF prevents DR by restructuring the microbiota toward species producing TUDCA and subsequent retinal protection by TGR5 activation.Item Type 2 diabetes disturbs Kir4.1 rhythm in retinal Müller cells(Office of the Vice Chancellor for Research, 2016-04-08) Luo, Qianyi; Bhatwadekar, AshayThe Müller cells function as a principal glia of the retina and maintain water homeostasis and K+ concentration via the specialized inwardly rectifying K+ (Kir) channels. About six to seven Kir channels have been found, among which Kir4.1 is expressed abundantly in Müller cells. Diabetes leads to a decrease in Kir4.1 expression and of potassium currents. For this study, we hypothesized that a diurnal change in Müller cell metabolism plays an important role in regulating the Kir4.1 expression. We tested our hypothesis using an animal model of type 2 diabetes (T2D;db/db mice) and in an in vitro study on the rat Müller (rMC-1) cells. The electroretinogram (ERG) assessment was performed on db/db mice to evaluate the Müller cell dysfunction. The rhythm of protein expression of Kir4.1 was examined in rMC-1 cells by western blot. The ‘b’ wave of an ERG, a characteristic of K+ ion distribution across the retina exhibited a diurnal rhythm in a mouse retina. The oscillatory pattern of ERG response was profoundly dampened in db/db mice. The clock synchronized rMC-1 cells in vitro exhibited a consistent oscillatory pattern for clock genes. The Kir4.1 protein in rMC-1 cells showed a regular pattern of the peak and troughs, consistent with the functional cock. Our studies suggest that Kir4.1 channels possess a diurnal rhythm and with T2D this rhythm is dampened.