A Cross-sectional Survey on Rodent Environmental Health Monitoring Practices: Benchmarking, Associations, and Barriers.

Tens of thousands of rodents are used each year in Rodent Health Monitoring programs. However, Environment Health Monitoring (EHM) could replace sentinel rodent use while maintaining or even improving diagnostic quality. Despite its advantages, widespread implementation of EHM appears to be relatively low. To better understand EHM's prevalence and factors influencing its use, we surveyed research animal professionals. Our hypotheses were (1) EHM prevalence would be low and (2) EHM use would be associated with beliefs and knowledge about EHM. Participants were recruited via online promotion. A total of 158 individuals completed a mixed-methods survey about current practices, beliefs, and knowledge about EHM. Qualitative data were coded using thematic analysis and analyzed using generalized linear models. Results showed that current EHM implementation was low; only 11% of institutions used EHM exclusively. Across the 111 institutions surveyed, over 20,000 soiled bedding sentinels were used each year. However, most participants believed EHM to be advantageous in replacing sentinel animals (78% of participants). Some participants believed EHM could save time (31%), cost less (27%), and be highly accurate (15%). Conversely, some participants believed EHM would be difficult to use due to their current caging type (40%), higher costs (21%), lower accuracy (16%), and personnel attitudes/expertise (14%). Overall, respondents with higher planned EHM use also had more positive attitudes, norms, and control of EHM. We also identified several factors that could promote the implementation of EHM. Communication efforts should emphasize that EHM is compatible with various types of caging, can provide cost savings, has high accuracy, and is consistent with the 3Rs as a replacement. Efforts should also focus on improving attitudes, encouraging peers, and providing resources to facilitate implementation. Implementation in just the surveyed institutions could eliminate the need for well over 20,000 rodents each year, consistent with 3Rs goals.

Increased MMP-9 levels with strain-dependent stress resilience and tunnel handling in mice.

Increased perineuronal net (PNN) deposition has been observed in association with corticosteroid administration and stress in rodent models of depression. PNNs are a specialized form of extracellular matrix (ECM) that may enhance GABA-mediated inhibitory neurotransmission to potentially restrict the excitation and plasticity of pyramidal glutamatergic neurons. In contrast, antidepressant administration increases levels of the PNN-degrading enzyme matrix metalloproteinase-9 (MMP-9), which enhances glutamatergic plasticity and neurotransmission. In the present study, we compare pro-MMP-9 levels and measures of stress in females from two mouse strains, C57BL/6 J and BALB/cJ, in the presence or absence of tail grasping versus tunnel-associated cage transfers. Prior work suggests that C57BL/6 J mice show relatively enhanced neuroplasticity and stress resilience, while BALB/c mice demonstrate enhanced susceptibility to adverse effects of stress. Herein we observe that as compared to the C57BL/6 J strain, BALB/c mice demonstrate a higher level of baseline anxiety as determined by elevated plus maze (EPM) testing. Moreover, as determined by open field testing, anxiety is differentially reduced in BALB/c mice by a choice-driven tunnel-entry cage transfer technique. Additionally, as compared to tail-handled C57BL/6 J mice, tail-handled BALB/c mice have reduced brain levels of pro-MMP-9 and increased levels of its endogenous inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1); however, tunnel-associated cage transfer increases pro-MMP-9 levels in BALB/c mice. BALB/c mice also show increases in Western blot immunoreactive bands for brevican, a constituent of PNNs. Together, these data support the possibility that MMP-9, an effector of PNN remodeling, contributes to the phenotype of strain and handling-associated differences in behavior.

Survey of One Health programs in U.S. medical schools and development of a novel one health elective for medical students.

Lessons learned from recent pandemics, such as SARS-CoV-2 have illustrated that education and training in a One Health approach, which recognizes the interdependency of the health of people, animals and the environment, are essential in improving preparations for and responses to disease outbreaks. For this reason and others, there is a critical need to provide One Health (OH) training to medical professionals early in their careers. 133 U.S. medical schools were surveyed for the incorporation of OH learning activities. Results showed that 56% of surveyed programs included OH-related subject matter, primarily in the context of preclinical classroom learning. This supports previous findings that OH education efforts in medical schools lag behind veterinary schools, with many veterinary schools already including OH as a central part of their curricula. A two week OH elective course for third year medical students was developed and implemented at Georgetown University School of Medicine. Topics such as emerging infectious diseases, zoonoses, vector-borne diseases, epidemiology, emergency preparedness, the human-animal bond, and effects of climate change on public health were discussed. The 21 participants were surveyed before and after the course regarding their knowledge and understanding of OH. Participation in the course enhanced the students' knowledge of OH and furthermore, the students' perception of the importance of incorporating OH within the curriculum and in their future careers changed significantly. This study provides clear evidence that successful integration of OH material is achievable at low cost through interdepartmental and interdisciplinary collaboration. A more holistic approach to health care that takes into consideration environmental, wildlife, and domestic animal factors, and introduction of concepts such as OH into the medical school curriculum, can help close the educational gaps identified in the surveys.

Clinical Management of Pain in Rodents.

The use of effective regimens for mitigating pain remain underutilized in research rodents despite the general acceptance of both the ethical imperative and regulatory requirements intended to maximize animal welfare. Factors contributing to this gap between the need for and the actual use of analgesia include lack of sufficient evidence-based data on effective regimens, under-dosing due to labor required to dose analgesics at appropriate intervals, concerns that the use of analgesics may impact study outcomes, and beliefs that rodents recover quickly from invasive procedures and as such do not need analgesics. Fundamentally, any discussion of clinical management of pain in rodents must recognize that nociceptive pathways and pain signaling mechanisms are highly conserved across mammalian species, and that central processing of pain is largely equivalent in rodents and other larger research species such as dogs, cats, or primates. Other obstacles to effective pain management in rodents have been the lack of objective, science-driven data on pain assessment, and the availability of appropriate pharmacological tools for pain mitigation. To address this deficit, we have reviewed and summarized the available publications on pain management in rats, mice and guinea pigs. Different drug classes and specific pharmacokinetic profiles, recommended dosages, and routes of administration are discussed, and updated recommendations are provided. Nonpharmacologic tools for increasing the comfort and wellbeing of research animals are also discussed. The potential adverse effects of analgesics are also reviewed. While gaps still exist in our understanding of clinical pain management in rodents, effective pharmacologic and nonpharmacologic strategies are available that can and should be used to provide analgesia while minimizing adverse effects. The key to effective clinical management of pain is thoughtful planning that incorporates study needs and veterinary guidance, knowledge of the pharmacokinetics and mechanisms of action of drugs being considered, careful attention to individual differences, and establishing an institutional culture that commits to pain management for all species as a central component of animal welfare.

Everything You Need to Know About Satisfying IACUC Protocol Requirements.

There have been recent efforts to reduce the administrative burden imposed on investigators. Although a complete and thorough review of proposed animal studies is an essential function of the Institutional Animal Care and Use Committee (IACUC), efforts to streamline and clarify this process may help investigators spend less time writing animal use protocols and responding to committee comments. The IACUC relies on well-written protocols for an efficient review process. A well-designed protocol form is also critical in guiding investigators through the process. However, it is ultimately the investigators' responsibility to ensure that the information they provide answers all the IACUC's questions with enough detail and quality for a fast and effective review. This article, aimed primarily for researchers but also IACUC administrators, provides an overview of the IACUC protocol review and approval process, the criteria that the IACUC uses for evaluations, and the type of information that should be included in the various sections of the protocol form. Some specific examples are also provided.

PCR Testing of Filter Material from IVC Lids for Microbial Monitoring of Mouse Colonies.

Testing sentinel animals exposed to soiled bedding from colony animals is the most common method used for health monitoring in rodent facilities. Although environmental sampling is being explored-and, in many cases, has been implemented-as an alternative, exhaust plenum sampling is not effective for all ventilated rack designs. This study evaluated PCR testing of filter paper from sentinel cages on ventilated racks. We hypothesized that testing filter paper from cages containing soiled bedding would be as effective as testing sentinel mice and that periodic shaking of cages would generate sufficient particulate movement to substitute for the presence of live animals. Three cages containing soiled bedding were maintained in each of 8 rooms; one cage contained 2 Cr:NIH(S) mice, one had no mice and was shaken twice weekly, and the remaining one had no mice and was left undisturbed. For 3 consecutive months, a piece of filter paper from the undersurface of the cage lid was tested monthly for adventitial agents and then replaced. A second piece remained on the cage undersurface for 3 mo. Fecal pellets and oral and fur swabs were collected from sentinel mice at months 1 and 3 and tested for the same agents. At month 3, serology was performed on the sentinel mice; feces and oral and fur swabs from colony animals were tested concurrently for comparison. Filter paper from cages without mice and shaken were at least as effective than all other methods in detecting the presence of endemic agents, including mouse norovirus, Helicobacter spp., Pasteurella pneumotropica, Entamoeba muris, and Spironucleus muris. For IVC systems where exhaust plenum testing is ineffective, PCR testing of IVC filter tops should be considered as an alternative to soiled bedding sentinels. Environmental sampling may provide increased reliability and reduce the number of rodents used for routine health surveillance.

Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets.

RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme.

Network Analysis Implicates Alpha-Synuclein (Snca) in the Regulation of Ovariectomy-Induced Bone Loss.

The postmenopausal period in women is associated with decreased circulating estrogen levels, which accelerate bone loss and increase the risk of fracture. Here, we gained novel insight into the molecular mechanisms mediating bone loss in ovariectomized (OVX) mice, a model of human menopause, using co-expression network analysis. Specifically, we generated a co-expression network consisting of 53 gene modules using expression profiles from intact and OVX mice from a panel of inbred strains. The expression of four modules was altered by OVX, including module 23 whose expression was decreased by OVX across all strains. Module 23 was enriched for genes involved in the response to oxidative stress, a process known to be involved in OVX-induced bone loss. Additionally, module 23 homologs were co-expressed in human bone marrow. Alpha synuclein (Snca) was one of the most highly connected "hub" genes in module 23. We characterized mice deficient in Snca and observed a 40% reduction in OVX-induced bone loss. Furthermore, protection was associated with the altered expression of specific network modules, including module 23. In summary, the results of this study suggest that Snca regulates bone network homeostasis and ovariectomy-induced bone loss.

Specificity and evolutionary conservation of the Escherichia coli RNA pyrophosphohydrolase RppH.

Bacterial RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5'-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5' end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5' terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution.

Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells.

In metazoans, cleavage by the endoribonuclease SMG6 is often the first degradative event in non-sense-mediated mRNA decay (NMD). However, the exact sites of SMG6 cleavage have yet to be determined for any endogenous targets, and most evidence as to the identity of SMG6 substrates is indirect. Here, we use Parallel Analysis of RNA Ends to specifically identify the 5' termini of decay intermediates whose production is dependent on SMG6 and the universal NMD factor UPF1. In this manner, the SMG6 cleavage sites in hundreds of endogenous NMD targets in human cells have been mapped at high resolution. In addition, a preferred sequence motif spanning most SMG6 cleavage sites has been discovered and validated by mutational analysis. For many SMG6 substrates, depletion of SMG6 resulted in the accumulation of decapped transcripts, an effect indicative of competition between SMG6-dependent and SMG6-independent NMD pathways. These findings provide key insights into the mechanisms by which mRNAs targeted by NMD are degraded.

Messenger RNA degradation in bacterial cells.

mRNA degradation is an important mechanism for controlling gene expression in bacterial cells. This process involves the orderly action of a battery of cellular endonucleases and exonucleases, some universal and others present only in certain species. These ribonucleases function with the assistance of ancillary enzymes that covalently modify the 5' or 3' end of RNA or unwind base-paired regions. Triggered by initiating events at either the 5' terminus or an internal site, mRNA decay occurs at diverse rates that are transcript specific and governed by RNA sequence and structure, translating ribosomes, and bound sRNAs or proteins. In response to environmental cues, bacteria are able to orchestrate widespread changes in mRNA lifetimes by modulating the concentration or specific activity of cellular ribonucleases or by unmasking the mRNA-degrading activity of cellular toxins.

Current options for providing sustained analgesia to laboratory animals.

The provision of adequate analgesia after an invasive procedure or for general pain management is an important component of laboratory animal care. Choosing the appropriate analgesic requires careful consideration by the investigators, the veterinary team and the institution's ethical review committee. Sustained-delivery analgesics are superior to analgesics with short durations of action because they do not need to be administered multiple times, reducing handling-induced stress to the animal, and they provide sustained plasma concentrations of the analgesic over the treatment period. The author reviews analgesic formulations that have durations of action longer than 12 h and up to 72 h. These options should be considered when appropriate for particular procedures and animal species.

Murine spinotrapezius model to assess the impact of arteriolar ligation on microvascular function and remodeling.

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published(1-3). For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 µm). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

A chitosan thermogel for delivery of ropivacaine in regional musculoskeletal anesthesia.

Postoperative pain within the first days following musculoskeletal surgeries is a significant problem for which appropriate management correlates to positive clinical outcomes. While a variety of pain management modalities are currently used for postoperative pain, an optimal strategy has yet to be identified. Utilizing local anesthetics to convey analgesia through neural blockade represents a promising approach to alleviate postoperative pain. Unfortunately, local anesthetics are often associated with short half-lives, local tissue site reactions, and systemic toxicity. Drug delivery systems such as liposomes, microparticles, and nanoparticles have been previously utilized to extend analgesia, but these systems can easily diffuse from the injection site. In order to overcome this limitation a combination of drug delivery technologies were utilized. Ropivacaine base nanoparticles were fabricated and entrapped with dexamethasone using a chitosan thermogel delivery system in order to enhance neural blockade. Using a rat sciatic neural blockade model, this system was able to limit sensory function and motor function for up to 48 h. This approach utilized a low solubility drug, a drug action enhancer, nanoparticles, and a thermogel matrix together to yield a multi-faceted delivery system capable of providing moderate-term pain management.

Differential control of the rate of 5'-end-dependent mRNA degradation in Escherichia coli.

Many Escherichia coli mRNAs are degraded by a 5'-end-dependent mechanism in which RppH-catalyzed conversion of the 5'-terminal triphosphate to a monophosphate triggers rapid endonucleolytic cleavage by RNase E. However, little is understood about what governs the decay rates of these transcripts. We investigated the decay of three such messages--rpsT P1, yfcZ, and ydfG--to characterize the rate-determining step in their degradation. The steady-state ratio of monophosphorylated to triphosphorylated rpsT P1 and yfcZ mRNA indicates that their decay rate is limited by cleavage of the monophosphorylated intermediate, making RNase E critical for their rapid turnover. Conversely, the decay rate of ydfG is limited by generation of the monophosphorylated intermediate; therefore, either RNase E or its less abundant paralog RNase G is sufficient for rapid ydfG degradation. Although all three transcripts are stabilized when RppH is absent, overproducing RppH does not accelerate their decay, nor does RppH overproduction appear to influence the longevity of most other messages that it targets. The failure of excess RppH to hasten rpsT P1 and yfcZ degradation despite increasing the percentage of each that is monophosphorylated is consistent with the observation that pyrophosphate removal is not the rate-limiting step in their decay. In contrast, neither the ydfG decay rate nor the fraction of ydfG transcripts that are monophosphorylated increases when the cellular concentration of RppH is raised, suggesting that, for some RppH targets, the rate of formation of the monophosphorylated intermediate is limited by an ancillary factor or by a step that precedes pyrophosphate removal.

Alignment and composition of laminin-polycaprolactone nanofiber blends enhance peripheral nerve regeneration.

Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin-polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits.

Fully covered self-expanding metal stents placed temporarily in the bile duct: safety profile and histologic classification in a porcine model.

BACKGROUND: Fully covered Self-Expanding metal stents (FCSEMS) have been shown efficacious in palliating malignant biliary obstructions. There is little data analyzing mucosal response to their temporary placement in the bile duct. METHODS: Ten mini pigs underwent endoscopic placement of a FCSEMS (Wallflex, Boston Scientific). FCSEMS were kept in place for three months. At the end of the 3 months, FCSEMS were removed endoscopically. Five pigs were euthanized and their bile ducts harvested. The other five were kept alive for another month post removal. A single pathologist, created a scoring system (to determine degree of inflammation, fibrosis, and epithelial injury), examined all specimens in a blinded fashion. RESULTS: Four FCSEMS spontaneously migrated in the duodenum. On post mortem examination, mild mucosal thickness was noted in three bile duct specimens while superficial inflammation of the bile duct was noted in five animals. Histologic examination of the bile duct revealed focal acute inflammation in both groups. For the 5 animals euthanized immediately after stent removal, there was a tendency to have superficial mucosal erosion and fibrosis. In contrast, increased chronic inflammation was more commonly seen in the animals 1 month post stent removal, with all animals in this group showing moderate degrees of mononuclear inflammatory cell mucosal infiltrates. No severe inflammatory or fibrotic duct injury was observed in any of the study animals, with degree of injury graded as mild to moderate. CONCLUSION: FCSEMS appear to induce minimal tissue overgrowth or fibrosis post placement. Ease of removability and no significant histologic injury are advantages noted with FCSEMS., however, further studies are needed to evaluate treating benign biliary strictures with FCSEMS in humans.

Evaluation of a sustained-release formulation of buprenorphine for analgesia in rats.

Preventing and minimizing pain in laboratory animals is a basic tenet of biomedical research and is warranted for ethical, legal, and scientific reasons. Postoperative analgesia is an important facet of pain management. A sustained-release formulation of buprenorphine was tested in rats for analgesic efficacy and plasma concentration over a 72-h time period. Rats were injected subcutaneously with either 1.2 mg/kg sustained-release formulation (Bup-SR), 0.2 mL/kg buprenorphine HCl (Bup-HCl), or an equivalent volume of sustained-release vehicle and tested in a thermal nociception model or a surgical postoperative pain model. In both models, Bup-SR showed evidence of providing analgesia for 2 to 3 d. Thermal latency response in rats that received the sustained-release formulation increased 28.4% and 15.6% compared with baseline values on days 1 and 2, respectively. Rats with a unicortical tibial defect and treated with Bup-SR showed similar willingness to bear weight on the hindlimbs as did negative-control animals (no surgery), demonstrated by counting vertical raises; rats treated with Bup-HCl had significantly fewer vertical raises than did control rats for 5 d after surgery. Plasma concentrations of buprenorphine remained over 1 ng/mL for 72 h after a single dose of Bup-SR. Taken together, the results indicate that this formulation of buprenorphine may be a viable option for treating postsurgical pain in laboratory rats.