Effects of slit lamp-delivered retinal laser photobiomodulation in a rat model of choroidal neovascularization.

Neovascular age-related macular degeneration, also known as exudative or wet age-related macular degeneration, is the leading cause of blindness in the developed world. Photobiomodulation has the potential to target the up-stream hypoxic and pro-inflammatory drivers of choroidal neovascularization. This study investigated whether photobiomodulation attenuates characteristic pathological features of choroidal neovascularization in a rodent model. Experimental choroidal neovascularization was induced in Brown Norway rats with laser photocoagulation. A custom-designed, slit-lamp-mounted, 670 nm laser was used to administer retinal photobiomodulation every 3 days, beginning 6 days prior to choroidal neovascularization induction and continuing until the animals were killed 14 days later. The effect of photobiomodulation on the size of choroidal neovascular membranes was determined using isolectin-B4 immunohistochemistry and spectral domain-optical coherence tomography. Vascular leakage was determined with fluorescein angiography. The effect of treatment on levels of vascular endothelial growth factor expression was quantified with enzyme-linked immunosorbent assay. Treatment with photobiomodulation was associated with choroidal neovascular membranes that were smaller, had less fluorescein leakage, and a diminished presence of inflammatory cells as compared to sham eyes. These effects were not associated with a statistically significant difference in the level of vascular endothelial growth factor when compared to sham eyes. The data shown herein indicate that photobiomodulation attenuates pathological features of choroidal neovascularization in a rodent model by mechanisms that may be independent of vascular endothelial growth factor.

Impact of processing on the sensitising capacity and cross-reactivity of cow's and camel milk proteins in a Brown Norway rat study.

Infant formulas based on hydrolysed cow's milk proteins are used when breastfeeding is not feasible in cow's milk allergic infants. Camel milk has been shown to be well-tolerated by the majority of children with cow's milk allergy (CMA) and may be a substitute in management of CMA. Here we aimed to evaluate the impact of processing on immunogenicity, sensitising, antibody-binding and cross-reactive capacity of cow's and camel milk. Cow's and camel milk were processed by means of enzyme hydrolysis or heat treatment. Brown Norway rats were immunised with PBS, non-processed, enzyme hydrolysed or heat-treated cow's or camel milk. In vivo tests were performed for evaluation of clinical signs. Blood and faecal samples were analysed for levels and specificity of antibody responses. Cow's and camel milk showed similar sensitising capacity. Processing decreased the sensitising capacity of cow's milk, yet only enzyme hydrolysis but not heat treatment decreased the sensitising capacity of camel milk. Processing affected the specificity of antibodies raised in the rats, though the effect differed between cow's and camel milk. The study showed a low cross-reactivity between cow's and camel milk, which was decreased with processing, suggesting that processing of camel milk may improve its usefulness in CMA management.

Stretch-shortening cycles protect against the age-related loss of power generation in rat single muscle fibres.

Aging is associated with impaired strength and power during isometric and shortening contractions, however, during lengthening (i.e., eccentric) contractions, strength is maintained. During daily movements, muscles undergo stretch-shortening cycles (SSCs). It is unclear whether the age-related maintenance of eccentric strength offsets age-related impairments in power generation during SSCs owing to the utilization of elastic energy or other cross-bridge based mechanisms. Here we investigated how aging influences SSC performance at the single muscle fibre level and whether performing active lengthening prior to shortening protects against age-related impairments in power generation. Single muscle fibres from the psoas major of young (∼8 months; n = 31 fibres) and old (∼32 months; n = 41 fibres) male F344BN rats were dissected and chemically permeabilized. Fibres were mounted between a force transducer and length controller and maximally activated (pCa 4.5). For SSCs, fibres were lengthened from average sarcomere lengths of 2.5 to 3.0 µm and immediately shortened back to 2.5 µm at both fast and slow (0.15 and 0.60 Lo/s) lengthening and shortening speeds. The magnitude of the SSC effect was calculated by comparing work and power during shortening to an active shortening contraction not preceded by active lengthening. Absolute isometric force was ∼37 % lower in old compared to young rat single muscle fibres, however, when normalized to cross-sectional area (CSA), there was no longer a significant difference in isometric force between age groups, meanwhile there was an ∼50 % reduction in absolute power in old as compared with young. We demonstrated that SSCs significantly increased power production (75-110 %) in both young and old fibres when shortening occurred at a fast speed and provided protection against power-loss with aging. Therefore, in older adults during everyday movements, power is likely 'protected' in part due to the stretch-shortening cycle as compared with isolated shortening contractions.

Topical Tacrolimus and Mycophenolic Acid Therapy Synergizes with Low Dose Systemic Immunosuppression to Sustain Vascularized Composite Allograft Survival.

AIM: The goal of this study was to evaluate whether topical administration of tacrolimus (TAC) and mycophenolic acid (MPA) at the transplant site enables vascularized composite allograft (VCA) survival with significant minimization of the dose and adverse effects of systemic TAC (STAC) immunosuppression. MATERIALS AND METHODS: Lewis (Lew) rats received orthotopic hind limb allotransplants from fully mismatched Brown Norway (BN) donors. Group 1 (Controls) received no treatment. Other groups were treated with STAC at a dose of 1 mg/kg/day for 7 days. On post-operative day (POD) 8, the STAC dose was dropped to 0.1 mg/kg/day for Group 2 and maintained at 1 mg/kg for Group 3. Group 4 received topical application of TAC and MPA on the transplanted (Tx) limb starting POD 8 without STAC. Group 5 received topical TAC and MPA on the contralateral non-Tx limb and Group 6 received topical TAC and MPA on the Tx limb starting POD 8 along with low dose STAC (0.1 mg/kg/day). Treatment was continued until the study end point was reached, defined as either grade 3 rejection or allograft survival exceeding 100 days. .We conducted sequential LC-MS/MS measurements to assess TAC and MPA concentrations in both blood/plasma and allograft tissues. Additionally, we evaluated markers indicative of organ toxicity associated with STAC immunosuppression. RESULTS: Compared to controls, topical therapy with TAC+MPA significantly prolonged allograft survival beyond 100 daysat very low dose STAC (0.1 mg/kg/day) (Group 6). The histopathological assessment of the grafts was consistent with the clinical outcomes. .Drug levels in blood/plasma remained low or undetectable, while allograft tissues showed higher drug concentrations compared to contralateral limb tissues (P<0.05). . Urinary creatinine clearance remained within the normal range at 2.5 mL/min. CONCLUSION: Combination therapy with topical TAC and MPA synergizes with a very low dose, corticosteroid- free-STAC regimen and facilitates rejection-free, prolonged VCA survival without morbidity.

Local Delivery of Adipose Stem Cells Promotes Allograft Survival in a Rat Hind-Limb Model of Vascularized Composite Allotransplantation.

BACKGROUND: Adipose stem cells (ASCs) are a promising cell-based immunotherapy because of their minimally invasive harvest, high yield, and immunomodulatory capacity. In this study, the authors investigated the effects of local versus systemic ASC delivery on vascularized composite allotransplant survival and alloimmune regulation. METHODS: Lewis rats received hind-limb transplants from Brown Norway rats and were administered donor-derived ASCs (passage 3 or 4, 1 × 10 6 cells/rat) locally in the allograft, or contralateral limb, or systemically at postoperative day 1. Recipients were treated intraperitoneally with rabbit anti-rat lymphocyte serum on postoperative days 1 and 4 and daily tacrolimus for 21 days. Limb allografts were monitored for clinical signs of rejection. Donor cell chimerism, immune cell differentiation, and cytokine expression in recipient lymphoid organs were measured by flow cytometric analysis. The immunomodulation function of ASCs was tested by mixed lymphocyte reaction assay and ASC stimulation studies. RESULTS: Local-ASC-treated recipients achieved significant prolonged allograft survival (85.7% survived >130 days; n = 6) compared with systemic-ASC and contralateral-ASC groups. Secondary donor skin allografts transplanted to the local-ASC long-term surviving recipients accepted permanently without additional immunosuppression. The increases in donor cell chimerism and regulatory T-cells were evident in blood and draining lymph nodes of the local-ASC group. Moreover, mixed lymphocyte reaction showed that ASCs inhibited donor-specific T-cell proliferation independent of direct ASC-T-cell contact. ASCs up-regulated antiinflammatory molecules in response to cytokine stimulation in vitro. CONCLUSION: Local delivery of ASCs promoted long-term survival and modulated alloimmune responses in a full major histocompatibility complex-mismatched vascularized composite allotransplantation model and was more effective than systemic administration. CLINICAL RELEVANCE STATEMENT: ASCs are a readily available and abundant source of therapeutic cells that could decrease the amount of systemic immunosuppression required to maintain limb and face allografts.

Spontaneous nonsense mutation in the tuftelin 1 gene is associated with abnormal hair appearance and amelioration of glucose and lipid metabolism in the rat.

Recently, we have identified a recessive mutation, an abnormal coat appearance in the BXH6 strain, a member of the HXB/BXH set of recombinant inbred (RI) strains. The RI strains were derived from the spontaneously hypertensive rat (SHR) and Brown Norway rat (BN-Lx) progenitors. Whole genome sequencing of the mutant rats identified the 195875980 G/A mutation in the tuftelin 1 (Tuft1) gene on chromosome 2, which resulted in a premature stop codon. Compared with wild-type BXH6 rats, BXH6-Tuft1 mutant rats exhibited lower body weight due to reduced visceral fat and ectopic fat accumulation in the liver and heart. Reduced adiposity was associated with decreased serum glucose and insulin and increased insulin-stimulated glycogenesis in skeletal muscle. In addition, mutant rats had lower serum monocyte chemoattractant protein-1 and leptin levels, indicative of reduced inflammation. Analysis of the liver proteome identified differentially expressed proteins from fatty acid metabolism and ß-oxidation, peroxisomes, carbohydrate metabolism, inflammation, and proteasome pathways. These results provide evidence for the important role of the Tuft1 gene in the regulation of lipid and glucose metabolism and suggest underlying molecular mechanisms.NEW & NOTEWORTHY A new spontaneous mutation, abnormal hair appearance in the rat, has been identified as a nonfunctional tuftelin 1 (Tuft1) gene. The pleiotropic effects of this mutation regulate glucose and lipid metabolism. Analysis of the liver proteome revealed possible molecular mechanisms for the metabolic effects of the Tuft1 gene.

Diabetes-Induced Changes of the Rat ERG in Relation to Hyperglycemia and Acidosis.

PURPOSE: To understand the mechanism of changes in the c-wave of the electroretinogram (ERG) in diabetic rats, and to explore how glucose manipulations affect the c-wave. METHODS: Vitreal ERGs were recorded in control and diabetic Long-Evans rats, 3-60 weeks after IP vehicle or streptozotocin. A few experiments were performed on Brown Norway rats. Voltage responses to current pulses were used to measure the transepithelial resistance of the retinal pigment epithelium (RPE). RESULTS: During development of diabetes the b-wave amplitude progressively decreased to about half of the initial amplitude after a year. In contrast, the c-wave was strongly affected from the very beginning (3 weeks) of diabetes. In control rats, the c-wave was cornea-positive at lower illuminations but was cornea-negative at higher (photopic) illumination. In diabetics, the whole amplitude-intensity curve was shifted toward negativity. The magnitude of this shift was markedly affected by acute glucose manipulations in diabetics but not in controls. Increased blood glucose made the c-wave more negative, and decreased blood glucose with insulin had the opposite effect. Experimentally induced acidification of the retina had a small effect that was different from diabetes, shifting the c-wave toward positivity, slightly in controls and more noticeably in diabetics. One reason for the significant negativity of the diabetic ERG was a decrease of the cornea-positive response of the RPE due to a decrease of the transepithelial resistance. CONCLUSIONS: The ERG c-wave is more negative in diabetics than in control animals, and is far more sensitive to changes in blood glucose. The increased negativity is largely if not entirely due to changes in the transepithelial resistance of the RPE, an electrical analog of the breakdown of the blood-retinal barrier observed in other studies. The sensitivity of the c-wave to glucose in diabetics may also be due to changes in transepithelial resistance.

Development of an Automated Electroretinography Analysis Approach.

Purpose: Electroretinography (ERG) is used to assess retinal function in ophthalmology clinics and animal models of ocular disease; however, analyzing ERG waveforms can be a time-intensive process with interobserver variability. We developed ERGAssist, an automated approach, to perform non-subjective and repeatable feature identification ("marking") of the ERG waveform. Methods: The automated approach denoised the recorded waveforms and then located the b-wave after applying a lowpass filter. If an a-wave was present, the lowpass filter wave was also used to help locate the a-wave, which was considered the initial large negative response after the flash stimuli. Oscillatory potentials (OPs) were found using a bandpass filter on the denoised waveform. We used two cohorts. One was a Coherence cohort that consisted of ERGs with eight dark-adapted and three light-adapted stimuli in Brown Norway rats (-6 to 1.5 log cd·s/m2). The Verification cohort consisted of control and diabetic (DM) Long Evans rats. We examined retinal function using a five-step dark-adapted protocol (-3 to 1.9 log cd·s/m2). Results: ERGAssist showed a strong correlation with manual markings of ERG features in our Coherence dataset, including the amplitudes (a-wave: r2 = 0.99; b-wave: r2 = 0.99; OP: r2 = 0.92) and implicit times (a-wave: r2 = 0.96; b-wave: r2 = 0.90; OP: r2 = 0.96). In the Verification cohort, both approaches detected differences between control and DM animals and found longer OP implicit times (P < 0.0001) in DM animals. Conclusions: These results provide verification of ERGAssist to identify features of the full-field ERG. Translational Relevance: This ERG analysis approach can increase the rigor of basic science studies designed to investigate retinal function using full-field ERG. To aid the community, we have developed an open-source graphical user interface (GUI) implementing the methods presented.

A workflow to visualize vertebrate eyes in 3D.

PURPOSE: To establish a workflow to visualize the surgical anatomy in 3D based on histological data of eyes of experimental animals for improving the planning of complex surgical procedures. METHODS: Four C57BL/6J wild-type(wt) mouse eyes, three Brown Norway rat eyes and four Chinchilla Bastard rabbit eyes were enucleated and processed for standard histology with serial sections and hematoxylin and eosin staining. Image stacks were processed to obtain a representation of the eye anatomy in 3D. In addition, virtual image stacks and 3D point clouds were generated by processing sagittal sections of eyes with stepwise 180° rotation and projection around the eye axis to construct a rotationally symmetric 3D model from one single sagittal section. RESULTS: Serial sections of whole eyes of mice, rats and rabbits showed significant artifacts interfering with a practical image stack generation and straightforward 3D reconstruction despite the application of image registration techniques. A workflow was established to obtain a 3D image of the eye based on virtual image stacks and point cloud generation by rotation of a single sagittal section of the eye around the symmetry axis. By analyzing the tissue shrinkage during histological processing true biometric reconstructions of the eyes were feasible making the resulting model usable for 3D modeling and simulation, e.g. for planning of complex surgical procedures in different species. CONCLUSION: Because serial sections of the eye with standard histological protocols yielded too many artifacts for a straightforward 3D visualization we reconstructed a pseudorealistic 3D model based on virtual image stacks and point cloud generation calculated from a single sagittal section of the eye. Such a model detailing microscopic structures of the whole eye will allow for a specific planning of surgical procedures in small animal eyes in order to prevent surgical complications in a very early stage of an experiment and it will support the design and development of complex intraocular implants. It will therefore be helpful in surgical teaching and improve laboratory animal welfare by an expected reduction of experimental animal numbers. Further processing including integration of mechanical tissue properties is needed to convert these 3D models into a practical virtual reality teaching and simulation platform for eyes of several species.

Luteolin attenuates acute liver allograft rejection in rats by inhibiting T cell proliferation and regulating T cell subsets.

Allograft rejection continues to be a significant cause of morbidity and graft failure for liver transplant recipients. Existing immunosuppressive regimens have many drawbacks, thus safe and effective long-term immunosuppressive regimens are still required. Luteolin (LUT), a natural component found in many plants, has a variety of biological and pharmacological effects and shows good anti-inflammatory activity in inflammatory and autoimmune diseases. Nevertheless, it remains unclear how it affects acute organ rejection after allogeneic transplantation. In this study, a rat liver transplantation model was constructed to investigate the effect of LUT on acute rejection of organ allografts. We found that LUT significantly protected the structure and function of liver grafts, prolonged recipient rat survival, ameliorated T cell infiltration, and downregulated proinflammatory cytokines. Moreover, LUT inhibited the proliferation of CD4+ T cells and Th cell differentiation but increased the proportion of Tregs, which is the key to its immunosuppressive effect. In vitro, LUT also significantly inhibited CD4+ T cell proliferation and Th1 differentiation. There may be important implications for improving immunosuppressive regimens for organ transplantation as a result of this discovery.

Descending Control of Nociception Poorly Predicts the Development of Persistent Postsurgical Pain-like Behavior in Consomic Dahl S Rat Strains.

BACKGROUND: Chronic postsurgical pain is a poorly recognized outcome of surgery where patients experience pain long after healing from the surgical insult. Descending control of nociception, a phenomenon whereby application of a strong nociceptive stimulus to one part of the body of animals inhibits pain in remote body regions, offers one strategy to identify a propensity to develop chronic postsurgical pain-like behavior. Here, consomic rat panel was used to test the hypothesis that pain persistence is mechanistically linked to ineffective descending control of nociception. METHODS: Male and female Brown Norway, Dahl S, and eight consomic strains (SS-xBN) were used to determine the presence of chronic postsurgical pain-like behaviors by using paw-withdrawal threshold evaluation (von Frey method) in the area adjacent to a hind paw plantar incision. Descending control of nociception was assessed by measuring hind paw-withdrawal thresholds (Randall-Selitto method) after capsaicin (125 µg) injection into a forepaw. Consomic rats were developed by introgressing individual Brown Norway chromosomes on the Dahl S rat genetic background, as Dahl S rats lack preoperative descending control of nociception. RESULTS: Substitution of several chromosomes from the "pain-resistant" Brown Norway to the "pain-prone" Dahl S/Medical College of Wisconsin reduced mechanical nociceptive sensitivity and increased endogenous pain modulation capacity by differing degrees. Statistical modeling of these data revealed that descending control of nociception is a poor general predictor of the propensity to develop chronic postsurgical pain-like behavior (poor fit for model 1). However, a significant strain-by-descending control of nociception interaction was revealed (model 3, -2*log likelihood; 550.668, -2ll change; 18.093, P = 0.034) with SS-13BN and SS-15BN strains showing a negative descending control of nociception relationship with chronic postsurgical pain-like behavior. CONCLUSIONS: Descending control of nociception poorly predicted which rat strains developed chronic postsurgical pain-like behavior despite controlling for genetic, environmental, and sex differences. Two consomic strains that mimic clinical chronic postsurgical pain criteria and display a strong negative correlation with descending control of nociception were identified, offering novel candidates for future experiments exploring mechanisms that lead to chronic postsurgical pain.

Adipose-Derived Mesenchymal Stem Cells Attenuate Immune Reactions Against Pig Decellularized Bronchi Engrafted into Rat Tracheal Defects.

Decellularized scaffolds are promising biomaterials for tissue and organ reconstruction; however, strategies to effectively suppress the host immune responses toward these implants, particularly those without chemical crosslinking, remain warranted. Administration of mesenchymal stem cells is effective against immune-mediated inflammatory disorders. Herein, we investigated the effect of isogeneic abdominal adipose-derived mesenchymal stem/stromal cells (ADMSCs) on xenogeneic biomaterial-induced immunoreactions. Peripheral bronchi from pigs, decellularized using a detergent enzymatic method, were engrafted onto tracheal defects of Brown Norway (BN) rats. BN rats were implanted with native pig bronchi (Xenograft group), decellularized pig bronchi (Decellularized Xenograft), or Decellularized Xenograft and ADMSCs (Decellularized Xenograft+ADMSC group). In the latter group, ADMSCs were injected intravenously immediately post implantation. Harvested graft implants were assessed histologically and immunohistochemically. We found that acute rejections were milder in the Decellularized Xenograft and Decellularized Xenograft+ADMSC groups than in the Xenograft group. Mild inflammatory cell infiltration and reduced collagen deposition were observed in the Decellularized Xenograft+ADMSC group. Additionally, ADMSC administration decreased CD8+ lymphocyte counts but increased CD163+ cell counts. In the Decellularized Xenograft+ADMSC group, serum levels of vascular endothelial growth factor and IL-10 were elevated and tissue deposition of IgM and IgG was low. The significant immunosuppressive effects of ADMSCs illustrate their potential use as immunosuppressive agents for xenogeneic biomaterial-based implants.

Longitudinal Observation of Retinal Response to Optic Nerve Transection in Rats Using Visible Light Optical Coherence Tomography.

Purpose: To characterize rat retinal responses after optic nerve transection (ONT) by visible-light optical coherence tomography (vis-OCT). Methods: Unilateral ONT was performed in Brown Norway rats (n = 8). In vivo, vis-OCT retinal imaging was performed on the experimental eyes before ONT (baseline), and two days, one week, two weeks, and four weeks (endpoint) after ONT, as well as on fellow eyes at the endpoint. The system was operated at a 70 kHz A-line sampling rate with both raster scans (512 × 2 × 512 A-lines), and circular scans (2048 × 100 A-lines) acquired around the optic disc. Retinal layers were segmented to calculate layer thicknesses and project en face images for visualization and quantifications. Vessel densities and oxygen saturation were used to evaluate the morphologic and functional impact on the retinal vasculature. Results: After ONT, retinal nerve fiber bundles demonstrated significant degeneration, starting at two weeks, with a reduction of thicknesses quantified on the nerve fiber layer, ganglion cell complex, and total retina. Along with that, the activation of macrophage-like cells in the vitreoretinal interface was also observed. Vessel densities for all three retinal plexuses were unaffected over the period of observation. However, oxygen saturation in retinal arteries and veins was significantly reduced at four weeks after ONT. Conclusions: Vis-OCT can provide high-definition, in vivo characterization of retinal responses to ONT in rats. Despite a significant reduction in retinal layer thickness, this was not accompanied by alterations in vascular density. Despite this, oximetry indicates reduced retinal oxygen saturation, suggesting that altered vascular physiology is not reflected in the anatomic appearance of retinal blood vessel density alone.

Dose and route of administration determine the efficacy of prophylactic immunotherapy for peanut allergy in a Brown Norway rat model.

Introduction: Allergen-specific immunotherapy (IT) is emerging as a viable option for treatment of peanut allergy. Yet, prophylactic IT remains unexplored despite early introduction of peanut in infancy was shown to prevent allergy. There is a need to understand how allergens interact with the immune system depending on the route of administration, and how different dosages of allergen may protect from sensitisation and a clinical active allergy. Here we compared peanut allergen delivery via the oral, sublingual (SL), intragastric (IG) and subcutaneous (SC) routes for the prevention of peanut allergy in Brown Norway (BN) rats. Methods: BN rats were administered PBS or three different doses of peanut protein extract (PPE) via either oral IT (OIT), SLIT, IGIT or SCIT followed by intraperitoneal (IP) injections of PPE to assess the protection from peanut sensitisation. The development of IgE and IgG1 responses to PPE and the major peanut allergens were evaluated by ELISAs. The clinical response to PPE was assessed by an ear swelling test (EST) and proliferation was assessed by stimulating splenocytes with PPE. Results: Low and medium dose OIT (1 and 10 mg) and all doses of SCIT (1, 10, 100 µg) induced sensitisation to PPE, whereas high dose OIT (100 mg), SLIT (10, 100 or 1000 µg) or IGIT (1, 10 and 100 mg) did not. High dose OIT and SLIT as well as high and medium dose IGIT prevented sensitisation from the following IP injections of PPE and suppressed PPE-specific IgE levels in a dose-dependent manner. Hence, administration of peanut protein via different routes confers different risks for sensitisation and protection from peanut allergy development. Overall, the IgE levels toward the individual major peanut allergens followed the PPE-specific IgE levels. Discussion: Collectively, this study showed that the preventive effect of allergen-specific IT is determined by the interplay between the specific site of PPE delivery for presentation to the immune system, and the allergen quantity, and that targeting and modulating tolerance mechanisms at specific mucosal sites may be a prophylactic strategy for prevention of peanut allergy.

Morphometric Analysis of Retinal Ganglion Cell Axons in Normal and Glaucomatous Brown Norway Rats Optic Nerves.

Purpose: A reference atlas of optic nerve (ON) retinal ganglion cell (RGC) axons could facilitate studies of neuro-ophthalmic diseases by detecting subtle RGC axonal changes. Here we construct an RGC axonal atlas for normotensive eyes in Brown Norway rats, widely used in glaucoma research, and also develop/evaluate several novel metrics of axonal damage in hypertensive eyes. Methods: Light micrographs of entire ON cross-sections from hypertensive and normotensive eyes were processed through a deep learning-based algorithm, AxoNet2.0, to determine axonal morphological properties and were semiquantitatively scored using the Morrison grading scale (MGS) to provide a damage score independent of AxoNet2.0 outcomes. To construct atlases, ONs were conformally mapped onto an ON "template," and axonal morphometric data was computed for each region. We also developed damage metrics based on myelin morphometry. Results: In normotensive eyes, average axon density was ∼0.3 axons/µm2 (i.e., ∼80,000 axons in an ON). We measured axoplasm diameter, eccentricity, cross-sectional area, and myelin g-ratio and thickness. Most morphological parameters exhibited a wide range of coefficients of variation (CoV); however, myelin thickness CoV was only ∼2% in normotensive eyes. In hypertensive eyes, increased myelin thickness correlated strongly with MGS (P < 0.0001). Conclusions: We present the first comprehensive normative RGC axon morphometric atlas for Brown Norway rat eyes. We suggest objective, repeatable damage metrics based on RGC axon myelin thickness for hypertensive eyes. Translational Relevance: These tools can evaluate regional changes in RGCs and overall levels of damage in glaucoma studies using Brown Norway rats.

Examination of the exposome in an animal model: The impact of high fat diet and rat strain on local and systemic immune markers following occupational welding fume exposure.

The goal of this study was to investigate the impact of multiple exposomal factors (genetics, lifestyle factors, environmental/occupational exposures) on pulmonary inflammation and corresponding alterations in local/systemic immune parameters. Accordingly, male Sprague-Dawley (SD) and Brown Norway (BN) rats were maintained on either regular (Reg) or high fat (HF) diets for 24wk. Welding fume (WF) exposure (inhalation) occurred between 7 and 12wk. Rats were euthanized at 7, 12, and 24wk to evaluate local and systemic immune markers corresponding to the baseline, exposure, and recovery phases of the study, respectively. At 7wk, HF-fed animals exhibited several immune alterations (blood leukocyte/neutrophil number, lymph node B-cell proportionality)-effects which were more pronounced in SD rats. Indices of lung injury/inflammation were elevated in all WF-exposed animals at 12wk; however, diet appeared to preferentially impact SD rats at this time point, as several inflammatory markers (lymph node cellularity, lung neutrophils) were further elevated in HF over Reg animals. Overall, SD rats exhibited the greatest capacity for recovery by 24wk. In BN rats, resolution of immune alterations was further compromised by HF diet, as many exposure-induced alterations in local/systemic immune markers were still evident in HF/WF animals at 24wk. Collectively, HF diet appeared to have a greater impact on global immune status and exposure-induced lung injury in SD rats, but a more pronounced effect on inflammation resolution in BN rats. These results illustrate the combined impact of genetic, lifestyle, and environmental factors in modulating immunological responsivity and emphasize the importance of the exposome in shaping biological responses.

Mutation in Smek2 regulating hepatic glucose metabolism causes hypersarcosinemia and hyperhomocysteinemia in rats.

Suppressor of mek1 (Dictyostelium) homolog 2 (Smek2), was identified as one of the responsible genes for diet-induced hypercholesterolemia (DIHC) of exogenously hypercholesterolemic (ExHC) rats. A deletion mutation in Smek2 leads to DIHC via impaired glycolysis in the livers of ExHC rats. The intracellular role of Smek2 remains obscure. We used microarrays to investigate Smek2 functions with ExHC and ExHC.BN-Dihc2BN congenic rats that harbor a non-pathological Smek2 allele from Brown-Norway rats on an ExHC background. Microarray analysis revealed that Smek2 dysfunction leads to extremely low sarcosine dehydrogenase (Sardh) expression in the liver of ExHC rats. Sarcosine dehydrogenase demethylates sarcosine, a byproduct of homocysteine metabolism. The ExHC rats with dysfunctional Sardh developed hypersarcosinemia and homocysteinemia, a risk factor for atherosclerosis, with or without dietary cholesterol. The mRNA expression of Bhmt, a homocysteine metabolic enzyme and the hepatic content of betaine (trimethylglycine), a methyl donor for homocysteine methylation were low in ExHC rats. Results suggest that homocysteine metabolism rendered fragile by a shortage of betaine results in homocysteinemia, and that Smek2 dysfunction causes abnormalities in sarcosine and homocysteine metabolism.

Aging affects gene expression in spermatids of Brown Norway rats.

The effects of aging on the reproductive health of men and the consequences for their offspring are becoming more widely recognized. Correlative epidemiological studies examining paternal age and offspring health suggest there are more frequent occurrences of genetic disorders in the children of older fathers. Given the genetic basis for paternal age-related disorders, we aim to characterize gene expression in developing germ cells. Round spermatids (RS) were collected from young (mean = 5.3 months) and aged (mean = 19.5 months) Brown Norway rats, representative of humans aged 20-30 years and 55+ years, respectively. Gene expression data were obtained by mRNA sequencing (n = 5), and were analysed for differential expression. Sequencing data display 211 upregulated and 9 downregulated transcripts in RS of aged rats, compared to young (log2FC >1, p < 0.05). Transcripts with increased expression are involved in several processes including sperm motility/morphology, sperm-egg binding, capacitation, and epigenetic inheritance. In addition, there are numerous dysregulated transcripts that regulate germ cell epigenetic marks and Sertoli-germ cell binding and communication. These results show an overall increase in RS gene expression with age, with spermatogenic functions being perturbed. Taken together, these findings help identify the genetic origin of the fertility, germ cell niche, and epigenetic effects observed with advanced paternal aging.

Reciprocal Donor-Recipient Strain Combinations Present Different Vascularized Composite Allotransplantation Outcomes in Rodent Models.

BACKGROUND: Although vascularized composite allotransplantation (VCA) has been the focus of many animal studies, further research is needed to determine the potential for a generalized model and immunosuppression regimen that applies across different donor-recipient combinations. In this study, the authors evaluated the outcome of VCAs performed on reciprocal rodent donor-recipient combinations. METHODS: VCA was performed in rats using Lewis and Brown Norway (BN) donor-recipient pairs, under the previously reported antilymphocyte serum/cyclosporine/adipose-derived stem cell regimen. Similarly, a published co-stimulatory blockade/rapamycin regimen was performed on the mouse VCA model between Balb/C and C57BL/6 strains. RESULTS: To accommodate the active behaviors of BN recipients, the allograft had to be modified and inset to the neck instead of to the groin. The tolerogenic regimen did not provide the same benefits for BN rats as it did for Lewis recipients. Increasing antilymphocyte serum dose and extending the duration of cyclosporine administration from 10 to 21 days significantly prolonged allograft survival and induced donor-specific tolerance. In mice, the co-stimulatory blockade/rapamycin regimen produced inferior VCA outcomes in BALB/c recipients than in C57BL/6 recipients. In both rats and mice, the authors identified an association between the tolerance outcome and the peripheral chimerism measured on postoperative day 30. CONCLUSIONS: Reciprocal donor-recipient combinations led to different responses toward the immunosuppression regimen and varied VCA outcomes. Sustained donor chimerism that remained in circulation for 1 month after surgery supported long-term VCA survival. Modification of the model and immunosuppression regimen accordingly is recommended. CLINICAL RELEVANCE STATEMENT: Various donor-recipient combinations respond differently to the immunosuppression regimens. Maintaining donor chimerism for 30 days after surgery improves VCA survival. It is recommended to tailor the immunosuppression regimen based on the recipient's background to optimize outcomes.

Licochalcone A alleviates laser-induced choroidal neovascularization by inhibiting the endothelial-mesenchymal transition via PI3K/AKT signaling pathway.

Choroidal neovascularization (CNV) is a hallmark of wet age-related macular degeneration, which severely impairs central vision. Studies have shown that endothelial-mesenchymal transition (EndMT) is involved in the pathogenesis of CNV. Licochalcone A (lico A), a flavonoid extracted from the root of licorice, shows the inhibition on EndMT, but it remains unclear whether it can suppress the formation of CNV. The aim of this study is to investigate the effects of lico A on laser-induced CNV, and EndMT process in vitro and vivo. We established the model of CNV with a krypton laser in Brown-Norway rats and then intraperitoneally injected lico A. Our experimental results demonstrated that the leakage of CNV was relieved, and the area of CNV was reduced in lico A-treated rats. Cell migration and tube formation in oxidized low-density lipoprotein (Ox-LDL)-stimulated HUVECs were inhibited by lico A and promoted by PI3K activator 740Y-P. The protein expressions of snai1 and α-SMA were increased, and CD31 and VE-cadherin were decreased in the model rats of CNV, but partially reversed after treatment with lico A. The expression of CD31 was decreased and α-SMA was increased in OX-LDL-treated HUVECs, which was further strengthened by 740Y-P, while the expression of CD31 was up-regulated and α-SMA was down-regulated in lico A treated HUVECs. Our data revealed that EndMT process was alleviated by lico A. Meanwhile, PI3K/AKT signaling pathway was activated in model rat of CNV and Ox-LDL-stimulated HUVECs, which can be suppressed with treatment of lico A. Our experimental results confirmed for the first time that lico A has the potential to alleviate CNV by inhibiting the endothelial-mesenchymal transition via PI3K/AKT signaling pathway.