Novel intravital approaches to quantify deep vascular structure and perfusion in the aging mouse brain using ultrasound localization microscopy (ULM).

Intra-vital visualization of deep cerebrovascular structures and blood flow in the aging brain has been a difficult challenge in the field of neurovascular research, especially when considering the key role played by the cerebrovasculature in the pathogenesis of both vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD). Traditional imaging methods face difficulties with the thicker skull of older brains, making high-resolution imaging and cerebral blood flow (CBF) assessment challenging. However, functional ultrasound (fUS) imaging, an emerging non-invasive technique, provides real-time CBF insights with notable spatial-temporal resolution. This study introduces an enhanced longitudinal fUS method for aging brains. Using elderly (24-month C57BL/6) mice, we detail replacing the skull with a polymethylpentene window for consistent fUS imaging over extended periods. Ultrasound localization mapping (ULM), involving the injection of a microbubble (<<10 µm) suspension allows for recording of high-resolution microvascular vessels and flows. ULM relies on the localization and tracking of single circulating microbubbles in the blood flow. A FIJI-based analysis interprets these high-quality ULM visuals. Testing on older mouse brains, our method successfully unveils intricate vascular specifics even in-depth, showcasing its utility for longitudinal studies that require ongoing evaluations of CBF and vascular aspects in aging-focused research.

Introducing Graduate Students in Biomedical Sciences to an Institutional Animal Research Program.

At the University of Oklahoma Health Sciences Center, graduate students enrolled in biomedical science programs comprise a significant portion of personnel working with research animals. Although the University requires that all personnel receive appropriate training before working with animals, veterinarians and research mentors agreed students would benefit from additional training. As a result, a course entitled "Laboratory Animal Use and Concepts" was added to the curriculum of the University's largest graduate program in biomedical sciences starting in 2017. The course introduces students to a number of topics relevant to the use of animals in biomedical research, with an emphasis on mice. Here we present a summary of the course and an assessment of its impact during the initial 5 y, from 2017-2021. Enrollment, student outcomes, and student evaluation surveys were included in this assessment. The course was offered to 6 classes totaling more than 120 students during this period. After completing the course, nearly 80% of students used animals as part of their graduate training. Among those, at least 21% sought additional training through formal workshops that offered supplemental opportunities to practice animal handling techniques. Student feedback suggested strong satisfaction with the course content and an appreciation for wet lab sessions. Providing enhanced training for incoming graduate students through this structured course appears to help students improve knowledge, skills, and attitudes that facilitate the responsible and ethical use of animals in biomedical research.

Evaluating IACUCs: Previous Research and Future Directions.

IACUCs serve a critical role in animal care and use programs, ensuring that institutions which use animals in research and teaching do so responsibly and humanely. This role is defined in part by federal regulations, policies, and guidelines that prescribe the establishment and function of these committees. Often, IACUC administrators are expected to evaluate IACUC performance to ensure that committees execute these functions effectively, and in a manner that is suitable to the institution. However, methods for IACUC performance evaluation have not been well described in the peer-reviewed literature. To address this deficit, we conducted a systematic review using MEDLINE to identify methods that have been used to assess IACUCs. The scope of this review was intentionally broad to capture evaluation methods used by other institutional committees with similar responsibilities in overseeing research conduct, including animal ethics committees (AECs), institutional biosafety committees (IBCs), and institutional review boards (IRBs). Over 100 publications that included empirical evaluation methods were identified, although only 17 evaluated IACUCs in the United States. A substantial number of the studies used qualitative methods, such as surveys or questionnaires, interviews, and observations. The IACUC functions and characteristics most often assessed in the 17 publications included components of the protocol review processes and committee membership. We compiled this information to offer IACUC administrators a source of methodologies that can be incorporated into quality improvement and IACUC performance evaluation efforts. We also suggest ways in which organizations may evaluate IACUCs using methods described in the literature for other types of committees.

PBN (Phenyl-N-Tert-Butylnitrone)-Derivatives Are Effective in Slowing the Visual Cycle and Rhodopsin Regeneration and in Protecting the Retina from Light-Induced Damage.

A2E and related toxic molecules are part of lipofuscin found in the retinal pigment epithelial (RPE) cells in eyes affected by Stargardt's disease, age-related macular degeneration (AMD), and other retinal degenerations. A novel therapeutic approach for treating such degenerations involves slowing down the visual cycle, which could reduce the amount of A2E in the RPE. This can be accomplished by inhibiting RPE65, which produces 11-cis-retinol from all-trans-retinyl esters. We recently showed that phenyl-N-tert-butylnitrone (PBN) inhibits RPE65 enzyme activity in RPE cells. In this study we show that like PBN, certain PBN-derivatives (PBNDs) such as 4-F-PBN, 4-CF3-PBN, 3,4-di-F-PBN, and 4-CH3-PBN can inhibit RPE65 and synthesis of 11-cis-retinol in in vitro assays using bovine RPE microsomes. We further demonstrate that systemic (intraperitoneal, IP) administration of these PBNDs protect the rat retina from light damage. Electroretinography (ERG) and histological analysis showed that rats treated with PBNDs retained ~90% of their photoreceptor cells compared to a complete loss of function and 90% loss of photoreceptors in the central retina in rats treated with vehicle/control injections. Topically applied PBN and PBNDs also significantly slowed the rate of the visual cycle in mouse and baboon eyes. One hour dark adaptation resulted in 75-80% recovery of bleachable rhodopsin in control/vehicle treated mice. Eye drops of 5% 4-CH3-PBN were most effective, inhibiting the regeneration of bleachable rhodopsin significantly (60% compared to vehicle control). In addition, a 10% concentration of PBN and 5% concentration of 4-CH3-PBN in baboon eyes inhibited the visual cycle by 60% and by 30%, respectively. We have identified a group of PBN related nitrones that can reach the target tissue (RPE) by systemic and topical application and slow the rate of rhodopsin regeneration and therefore the visual cycle in mouse and baboon eyes. PBNDs can also protect the rat retina from light damage. There is potential in developing these compounds as preventative therapeutics for the treatment of human retinal degenerations in which the accumulation of lipofuscin may be pathogenic.

Evaluation of reproduction and raising offspring in a nursery-reared SPF baboon (Papio hamadryas anubis) colony.

Baboons (Papio hamadryas anubis) of a conventional breeding colony were nursery-reared to create a specific pathogen-free (SPF) baboon-breeding program. Because the founding generations were nursery-reared until 2 years of age, it was suspected that the SPF baboons would exhibit increased reproductive challenges as adults. Mothering behavior was of interest, because SPF females were not exposed to parental role models during the nursery-rearing process. We compared reproductive data from the SPF baboon breeding program during its first 10 years with data from age-matched baboons during the same period from an established, genetically-similar conventional breeding colony. We also evaluated records documenting mother-infant behaviors within the SPF colony. The average age of menarche in SPF females was 3.3 years. The overall live birth rate of both SPF and conventional females was approximately 90%, with no difference in pregnancy outcome between the two colonies. The average age at first conception for SPF females was earlier (4.2 years) than that of the conventional females (4.7 years). In both colonies, primiparous females were more likely to abort than multiparous females. Similarly, primiparous females were more likely to lose their infants to death or human intervention. A mothering score system was developed in the SPF colony to facilitate intervention of poor mother-infant relationships. Records revealed 70% of SPF mothers were able to raise one or more of their infants successfully to at least 180 days of age, which did not differ from conventional mothers. SPF females returned to post-partum amenorrhea 27 days sooner on average than the conventional females, independent of dam age. The nursery-rearing process used for recruitment into the SPF colony therefore did not have an adverse effect on reproduction or rearing offspring.