Reprogramming to recover youthful epigenetic information and restore vision.

Ageing is a degenerative process that leads to tissue dysfunction and death. A proposed cause of ageing is the accumulation of epigenetic noise that disrupts gene expression patterns, leading to decreases in tissue function and regenerative capacity1-3. Changes to DNA methylation patterns over time form the basis of ageing clocks4, but whether older individuals retain the information needed to restore these patterns-and, if so, whether this could improve tissue function-is not known. Over time, the central nervous system (CNS) loses function and regenerative capacity5-7. Using the eye as a model CNS tissue, here we show that ectopic expression of Oct4 (also known as Pou5f1), Sox2 and Klf4 genes (OSK) in mouse retinal ganglion cells restores youthful DNA methylation patterns and transcriptomes, promotes axon regeneration after injury, and reverses vision loss in a mouse model of glaucoma and in aged mice. The beneficial effects of OSK-induced reprogramming in axon regeneration and vision require the DNA demethylases TET1 and TET2. These data indicate that mammalian tissues retain a record of youthful epigenetic information-encoded in part by DNA methylation-that can be accessed to improve tissue function and promote regeneration in vivo.

Microglia preserve visual function loss in the aging retina by supporting retinal pigment epithelial health.

BACKGROUND: Increased age is a risk factor for the development and progression of retinal diseases including age-related macular degeneration (AMD). Understanding the changes that occur in the eye due to aging is important in enhancing our understanding of AMD pathogenesis and the development of novel AMD therapies. Microglia, the resident brain and retinal immune cells are associated with both maintaining homeostasis and protection of neurons and loss of microglia homeostasis could be a significant player in age related neurodegeneration. One important characteristic of retinal aging is the migration of microglia from the inner to outer retina where they reside in the subretinal space (SRS) in contact with the retinal pigment epithelial (RPE) cells. The role of aged subretinal microglia is unknown. Here, we depleted microglia in aged C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony-stimulating factor-1 receptor (Csf1r) required for microglial survival. RESULTS: The subretinal P2RY12 + microglia in aged mice displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets reminiscent of lipofuscin. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, one of the main functions of retinal pigmented epithelial cells. PLX5622 treatment depleted up to 90% of the retinal microglia and was associated with significant loss in visual function. Mice on the microglia depletion diet showed reduced contrast sensitivity and significantly lower electroretinogram for the c-wave, a measurement of RPE functionality, compared to age-matched controls. The loss of c-wave coincided with a loss of RPE cells and increased RPE swelling in the absence of microglia. CONCLUSIONS: We conclude that microglia preserve visual function in aged mice and support RPE cell function, by phagocytosing shed photoreceptor outer segments and lipids, therefore compensating for the known age-related decline of RPE phagocytosis.

A More Comfortable Method for Hydrostatic Weighing: Head above Water at Total Lung Capacity.

Hydrostatic weighing (HW) requires full submersion with the lungs at residual volume (RV) which is uncomfortable. Therefore, the purpose of this study was to find a more comfortable way to complete HW. A HW system was used to complete three comparisons: comparison 1: change in head position (head above water vs. head below water (HAW vs. HBW)), comparison 2: change in lung volume (total lung capacity (TLC) vs. RV), and comparison 3: change in head and lung volume changes. Participants were separated by males (n = 64) and females (n = 58). Comparison 1: HAW resulted in higher mean percent body fat (PBF) than HBW (4.5% overall, 3.8% in males, 5.4% in females, p < 0.05). Comparison 2: TLC resulted in lower mean PBF than RV (5.1% overall, 5.3% in males, 4.8% in females, p < 0.05). Comparison 3: HAW@TLC resulted in significantly lower (1.5% lower, p = 0.003) mean PBF for males but was not significantly lower for females or overall (0.6% higher, p = 0.39, 0.6% lower, p = 0.18, respectively) compared to HBW@RV. In conclusion, keeping the head above water and taking a deep inhale makes HW a more enjoyable, and accessible experience for everyone while still producing accurate PBF results.

Effects of peripheral active warming and passive insulation on core body temperature during feline ovariohysterectomy: a multi-arm randomized clinical trial.

OBJECTIVES: Hypothermia is a common complication of anesthesia, particularly in cats. Some veterinarians insulate the extremities of cats as a preventive measure, and there is evidence that heating the extremities of dogs decreases the rate of heat loss from the core. This study investigated whether active warming or passive insulation of the extremities of cats resulted in a slower decrease in rectal temperature during anesthesia. METHODS: Female cats were assigned via block randomization to passive (cotton toddler socks), active (heated toddler socks) or control group (uncovered extremities). Rectal temperature was monitored every 5 mins from induction through return to trap/carrier (final temperature). Multivariable linear regression models were used to compare temperature (rate change and final) between groups. RESULTS: There were 164 cats with 1757 temperature readings. Mean total duration of anesthesia was 53 ± 13 mins. The temperature of all groups decreased linearly over time (all P <0.0001), with the rates of temperature decrease being -0.039°F/min (95% confidence interval [CI] -0.043 to -0.035)/-0.022°C (95% CI -0.024 to -0.019), -0.039°F/min (95% CI -0.042 to -0.035)/-0.022°C (95% CI -0.023 to -0.019) and -0.029°F/min (95% CI -0.032 to -0.025)/-0.016°C (95% CI -0.018 to -0.014) for the control, passive and active groups, respectively. The control, passive and active groups had median final temperatures of 98.4°F (interquartile range [IQR] 97.6-99.4)/36.9°C (IQR 36.4-37.4), 98.0°F (IQR 97.2-98.7)/36.7°C (IQR 36.2-37.1) and 99.1°F (IQR 97.7-100.0)/37.3°C (IQR 36.5-37.8), respectively. After controlling for weight, postinduction temperature and duration of anesthesia, and as compared with controls, the final temperature of the active group was predicted to be 0.54°F (95% CI 0.03-1.01)/0.3°C (95% CI 0.02-0.56) greater (P = 0.023), while the passive group was not significantly different (P = 0.130). CONCLUSIONS AND RELEVANCE: The rate of rectal temperature decrease was significantly slower for the active group compared with the other groups. Although the cumulative difference in final temperature reading was modest, superior materials might enhance performance. Cotton toddler socks alone did not slow the rate of temperature decrease.

Sustained Vision Recovery by OSK Gene Therapy in a Mouse Model of Glaucoma.

Glaucoma, a chronic neurodegenerative disease, is a leading cause of age-related blindness worldwide and characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons. Previously, we developed a novel epigenetic rejuvenation therapy, based on the expression of the three transcription factors Oct4, Sox2, and Klf4 (OSK), which safely rejuvenates RGCs without altering cell identity in glaucomatous and old mice after 1 month of treatment. In the current year-long study, mice with continuous or cyclic OSK expression induced after glaucoma-induced vision damage had occurred were tracked for efficacy, duration, and safety. Surprisingly, only 2 months of OSK fully restored impaired vision, with a restoration of vision for 11 months with prolonged expression. In RGCs, transcription from the doxycycline (DOX)-inducible Tet-On AAV system, returned to baseline 4 weeks after DOX withdrawal. Significant vision improvements remained for 1 month post switching off OSK, after which the vision benefit gradually diminished but remained better than baseline. Notably, no adverse effects on retinal structure or body weight were observed in glaucomatous mice with OSK continuously expressed for 21 months providing compelling evidence of efficacy and safety. This work highlights the tremendous therapeutic potential of rejuvenating gene therapies using OSK, not only for glaucoma but also for other ocular and systemic injuries and age-related diseases.

The characteristics and clinical course of patients with COVID-19 who received invasive mechanical ventilation in Osaka, Japan.

OBJECTIVE: To describe the detailed clinical course of patients with coronavirus disease 2019 (COVID-19) who received invasive mechanical ventilation. METHODS: We conducted a case series of patients with COVID-19 who received invasive mechanical ventilation in Osaka, Japan, between January 29 and May 28, 2020. We describe the patient characteristics and clinical course from onset. Additionally, we fitted logistic regression models to investigate the associations between patient characteristics and the 30-day mortality rate. RESULTS: A total of 125 patients who received invasive mechanical ventilation (median age [interquartile range], 68 [57-73] years; male, 77.6%) were enrolled. Overall, the 30-day mortality was 24.0%, and the median (interquartile range) length of ICU stay and length of invasive mechanical ventilation use were 16 (12-29) days and 13 (9-26) days, respectively. From clinical onset, 121 patients (96.8%) were intubated within 14 days. In multivariable logistic regression analysis, age of 65 years or older (odds ratio, 3.56; 95% confidence interval, 1.21-10.49; P = 0.02) and male sex (odds ratio, 3.75; 95% confidence interval, 1.00-11.24, P = 0.04) were significantly associated with a higher 30-day mortality rate. CONCLUSIONS: In this case series of patients with COVID-19 who received invasive mechanical ventilation in Japan, the 30-day mortality rate was 24.0%, and age 65 years or older and male sex were associated with higher 30-day mortality rate.