Genus-group- and species-specific primers were used as described previously to quantify the different bacterial groups of the intestinal microbiota

Panx1 responsive to mechanical stimulation, extracellular purines, high extracellular K+, and other stimuli. A more recent study showed proteolytic activation of Panx1 by caspase-3 digestion and indicated that the channel plays an essential role in phagocyte attraction during apoptosis. Currently, the normal physiological function of Panx1 remains poorly understood. It was shown that cell swelling and membrane breakdown after ischemic injury are blocked by Lonafarnib price hemichannel inhibitors in pyramidal neurons, which express Panx1 but not connexins. These data, together with the findings that Panx1 channels are opened by extracellular ATP, nitric oxide and glutamate, suggested that Panx1 activation facilitates neurotoxicity in ischemic brain. Panx1 is also involved in the activation of a cytoplasmic protein complex known as the inflammasome. The inflammasome mediates proteolytic activation of caspases-1, which is a critical step in processing and secretion of pro-inflammatory cytokines IL1b, IL-18 and IL-33 in monocytes, astrocytes, as well as brain neurons. Over-production of IL-1b was shown to play Pannexin1 in Retinal Ischemia deleterious role in the central nervous system and inflammasome activation is now being implicated in multiple neurological conditions, including brain and spinal cord injury. The IL-1b toxicity can be suppressed by interleukin-1 receptor blockade, which alleviated damage in retinal ischemia model. Anti-IL-1b therapy is now a clinically proven therapy of autoinflammatory diseases, familial hereditary fever, gout, rheumatoid arthritis and type 2 diabetes mellitus and is in clinical trials for stroke patients. Equally efficient neuroprotection is achieved by alternative strategy, i.e. by direct blockade of inflammasome, as shown in rodent models of stroke and traumatic brain injury. The exact nature of signal leading to inflammasome activation in the CNS remains poorly understood. Among the mechanisms suggested recently is Panx1 channel-mediated internalization of external danger signals and Panx1-mediated activation of caspase-1. However, the connection between neuronal Panx1 channel and molecular underpinnings of ischemic degeneration of neurons remains PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189214 to be investigated. The overall aim of this study is to examine the role of Panx1 channels in the pathophysiology of retinal IR injury in vivo. The hemichannel blockers such as carbenoxolone, lanthanum and mefloquine that were commonly utilized to study the function of Panx1 in cultured cells are not suitable for this purpose. These chemicals are rather non-specific as they also inhibit connexons and stimulate unrelated pathways. To circumvent potential nonspecific effects commonly associated with pharmacological inhibitors, we developed a conditional Panx1 knockout mouse. This mouse model was instrumental to study the relationship between Panx1 activity and pathophysiology of ischemic loss of RGCs in the retina. We report here that Panx1 deficiency protects RGCs from death induced by ischemia. Our data provide evidence that Panx1 is involved in at least two toxicity mechanisms and suggest that Panx1 is a convergence point for several neurotoxic pathways and a new target for therapeutic intervention in retinal ischemic disorders. Results Panx1 conditional knockout mouse line To study the role of Panx1 channels in retinal ischemia we generated homozygous Panx1fl/fl mutant mice, harboring three LoxP consensus sites inserted into a single-copy Panx1 gene. Founders with germl