Breed-Specific Skull Morphology Reveals Insights into Canine Optic Chiasm Positioning and Orbital Structure through 3D CT Scan Analysis.

This study's CT scan-based morphometric analysis of 50 adult dogs explored the relationship between skull shape variations (determined by the skull index, SI), optic chiasm, optic canals, and orbital shape. Dogs were classified as brachycephalic (SI ≥ 59), mesocephalic (SI ≥ 51 but <59), and dolichocephalic (SI < 51). No significant age or weight differences were observed. Skull lengths (brachycephalic: 11.39 ± 1.76 cm, mesocephalic: 15.00 ± 2.96 cm, dolichocephalic: 17.96 ± 3.44 cm) and facial lengths (brachycephalic: 3.63 ± 1.00 cm, mesocephalic: 6.46 ± 1.55 cm, dolichocephalic: 8.23 ± 1.03 cm) varied significantly, with shorter orbital depths (brachycephalic: 2.58 ± 0.42 cm, mesocephalic: 3.19 ± 0.65 cm, dolichocephalic: 3.61 ± 0.77 cm) in brachycephalic dogs. The optic chiasm-to-inion horizontal length ratio to cranial horizontal length positively correlated with the SI (r = 0.883, p < 0.001), while the ratio to neurocranial length showed no SI correlation (range: 55.5-75.0). Brachycephalic breeds had a significantly wider optic canal angle (93.74 ± 16.00°), along with broader lacrimal-zygomatic and zygomatic frontal process angles. These findings highlight the zygomatic bone's role in influencing breed-specific orbital variations by connecting the face to the neurocranium, projecting the orbital rim outward and forward with facial shortening.

Oral 5-aminolevulinic acid combined with sodium ferrous citrate prevents blood-aqueous barrier breakdown after anterior chamber paracentesis in healthy beagle dogs.

This study investigated the preventive effect of 5-aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) on blood-aqueous barrier (BAB) breakdown induced after anterior chamber paracentesis (ACP) in beagles. 5-ALA/SFC (1/0.64 mg/kg or 3/1.92 mg/kg) or carprofen (4.0 mg/kg) was orally administered daily for 7 days prior to ACP. Then, a sample of the aqueous humor (AH) was collected from one eye via ACP (first sample) and again 60 min later (second sample). The protein and prostaglandin E2 (PGE2) concentrations in both samples were measured. Compared with the control group, high-dose 5-ALA/SFC and carprofen significantly reduced the AH protein and PGE2 concentrations in the second sample. Our findings suggest that 5-ALA/SFC suppresses BAB breakdown in dogs.

Nasal Absorption Enhancement of Mometasone Furoate Nanocrystal Dispersions.

Purpose: We designed a 0.05% mometasone furoate (MF) nanocrystal dispersion and investigated whether the application of MF nanocrystals in nasal formulations enhanced local absorption compared to traditional nasal MF formulations (CA-MF). Methods: MF nanocrystal dispersions (MF-NPs) were prepared by bead milling MF microcrystal dispersions (MF-MPs) consisting of MF, 2-hydroxypropyl-ß-cyclodextrin, methylcellulose, and purified water. Pluronic F-127 combined with methylcellulose, Pluronic F-68, or carbopol was used as a base for in situ gelation (thickener). MF concentrations were measured using high-performance liquid chromatography, and nasal absorption of MF was evaluated in 6 week-old male Institute of Cancer Research (ICR) mice. Results: The particle size range of MF prepared with the bead mill treatment was 80-200 nm, and the nanoparticles increased the local absorption of MF, which was higher than that of CA-MF and MF-MPs. In addition, unlike the results obtained in the small intestine and corneal tissue, the high absorption of nanocrystalline MF in the nasal mucosa was related to a pathway that was not derived from energy-dependent endocytosis. Moreover, the application of the in situ gelling system attenuated the local absorption of MF-NPs, owing to a decrease in drug diffusion in the dispersions. Conclusion: We found that nanoparticulation of MF enhances local intranasal absorption, and nasal bioavailability is higher than that of CA-MF. In addition, we demonstrate that viscosity regulation is an important factor in the design of nasal formulations based on MF nanocrystals. These findings provide insights for the design of novel nanomedicines with enhanced nasal bioavailability.

Sodium Ferrous Citrate and 5-Aminolevulinic Acid Exert a Therapeutic Effect on Endotoxin-Induced Uveitis in Rats.

The metabolism of 5-aminolevulinic acid (ALA) is more efficient when combined with sodium ferrous citrate (SFC). Our previous study revealed that oral administration of ALA, which has anti-inflammatory properties, and SFC (ALA/SFC) immediately before lipopolysaccharide (LPS) inoculation suppressed endotoxin-induced uveitis (EIU) in rats. However, the therapeutic effect of ALA/SFC post-administration remains unexplored. Hence, this study aimed to evaluate the therapeutic efficacy of ALA/SFC on EIU in rats, which were administered with a gastric gavage of ALA/SFC (100/157 mg/kg) or prednisolone (Pred, 10 mg/kg) after 4 h of LPS inoculation. The treatment groups showed ameliorated clinical scores, inflammatory cells, protein levels in the aqueous humor (AqH), and histopathologic evaluation 24 h after LPS inoculation. Furthermore, the treatment groups had reduced tumor necrosis factor-α, nitric oxide, prostaglandin E2, and interleukin-6 levels in the AqH. ALA/SFC demonstrated an anti-inflammatory effect equivalent to that demonstrated by Pred. These findings indicate that ALA/SFC exerts a therapeutic effect on EIU in rats, indicating its clinical usefulness in uveitis treatment.

5-ALA/SFC Ameliorates Endotoxin-Induced Ocular Inflammation in Rats by Inhibiting the NF-κB Signaling Pathway and Activating the HO-1/Nrf2 Signaling Pathway.

Sodium ferrous citrate (SFC) is involved in the metabolism of 5-aminolevulinic acid (5-ALA) and enhances its anti-inflammatory effects. The effects of 5-ALA/SFC on inflammation in rats with endotoxin-induced uveitis (EIU) have yet to be elucidated. In this study, during lipopolysaccharide injection, 5-ALA/SFC (10 mg/kg 5-ALA plus 15.7 mg/kg SFC) or 5-ALA (10 or 100 mg/kg) was administered via gastric gavage, wherein we saw that 5-ALA/SFC ameliorated ocular inflammation in EIU rats by suppressing clinical scores; by infiltrating cell counts, aqueous humor protein, and inflammatory cytokine levels; and by improving histopathological scores to the same extent as 100 mg/kg 5-ALA. Immunohistochemistry showed that 5-ALA/SFC suppressed iNOS and COX-2 expression, NF-κB activation, IκB-α degradation, and p-IKKα/ß expression, and activated HO-1 and Nrf2 expression. Therefore, this study has investigated how 5-ALA/SFC reduces inflammation and revealed the pathways involved in EIU rats. 5-ALA/SFC is shown to inhibit ocular inflammation in EIU rats by inhibiting NF-κB and activating the HO-1/Nrf2 pathways.

Effects of Oral 5-Aminolevulinic Acid on Lipopolysaccharide-Induced Ocular Inflammation in Rats.

This study aimed to investigate the anti-inflammatory effect of 5-aminolevulinic acid (5-ALA) on endotoxin-induced uveitis (EIU) in rats. EIU was induced in male Sprague Dawley rats by the subcutaneous injection of lipopolysaccharide (LPS). During LPS injection, 5-ALA diluted with saline was administered via gastric gavage. After 24 h, clinical scores were assessed after which aqueous humor (AqH) samples were obtained. The number of infiltrating cells, protein concentration, and levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) in AqH were measured. For histological examination, both eyes of some rats were enucleated. In vitro, a mouse macrophage cell line (RAW264.7 cells) was stimulated by LPS with or without 5-ALA. Western blot was used to analyze the expression of inducible NO synthase (iNOS) and cyclooxygenase-2. 5-ALA suppressed the EIU clinical scores, infiltrating cell number, and protein concentration while improving the histopathologic scores. In particular, 100 mg/kg 5-ALA reduced the concentrations of NO, PGE2, TNF-α, and IL-6 in AqH, similar to 1 mg/kg prednisolone. In addition, 5-ALA suppressed iNOS upregulation in LPS-stimulated RAW264.7 cells. Therefore, 5-ALA has an anti-inflammatory effect on EIU through the inhibition of the upregulation of inflammatory mediators.

Retention, Bacterial Adhesion, and Biofilm Formation between Anionic and Zwitterionic Bandage Contact Lenses in Healthy Dogs: A Pilot Study.

This study aimed to compare the in vitro and in vivo retention, bacterial adhesion, and biofilm formation between anionic and zwitterionic bandage contact lenses (BCLs) in healthy canines. BCL retention and tolerance were evaluated in 10 healthy canines via a single-masked, crossover study for 7 days. To compare in vitro bacterial adhesion and biofilm formation, four Staphylococcus strains were incubated with the BCLs at 37 °C for 2 or 24 h, and the bacterial colony forming units (CFUs) adhering to the BCLs were counted. Next, to compare in vivo bacterial adhesion, the CFUs of bacteria adhering to the BCLs worn by canines for 24 h were counted. Anionic lenses significantly retained and reduced in vitro bacterial adhesion than in the zwitterionic lenses. However, the amount of in vitro biofilm formation was more likely to be higher on anionic lenses than on zwitterionic lenses. In vivo bacterial adhesion was not significantly different between the two types of BCLs. Nevertheless, both BCLs were well-tolerated by the canines; thus, their short-term use in dogs can be recommended as safe.

Primary cell culture of canine corneal endothelial cells.

OBJECTIVE: To establish a primary cell culture and clarify the characteristics of canine corneal endothelial cells in vitro. PROCEDURES: The eyes were enucleated from dogs that were euthanized for reasons unrelated to this study. Enucleated canine eyes were dissected, and the intact corneas were isolated from the globes. Using enzymes, the corneal endothelial cells were dispersed from the cornea. The obtained canine corneal endothelial cells were cultured in a cell culture dish. Cultured corneal endothelial cells were morphologically evaluated using phase-contrast microscopy. Immunohistochemical analysis of the cultured cells, particularly of the corneal endothelial cell marker, zonula occludens-1 (ZO-1), Na+ /K+ -ATPase, and vimentin, was performed to clarify whether the cultured cells were actually corneal endothelial cells. Furthermore, the post-passage morphology of cultured cells was evaluated. RESULTS: Canine primary cultured corneal endothelial cells showed morphologically small, cobblestone-like structures. The isolated cells had proliferative ability in vitro and demonstrated positive expression of the corneal endothelial cell markers, ZO-1, Na+ /K+ -ATPase, and vimentin. However, repeated passages resulted in larger cell sizes as assessed by phase-contrast microscopy. Repeated passages also resulted in lower cell density. CONCLUSIONS: This study demonstrated the successful culture of canine corneal endothelial cells. This might enhance the understanding of corneal endothelial cell characteristics in dogs.

Magnesium Hydroxide Nanoparticles Improve the Ocular Hypotensive Effect of Twice Daily Topical Timolol Maleate in Healthy Dogs.

Timolol maleate (TM), a beta-adrenergic receptor antagonist, is widely used for canine antiglaucoma eye drops; however, its bioavailability is <5%. Our previous study revealed that magnesium hydroxide nanoparticles (nMH) have potency in improving the bioavailability of fixed-combined TM in rodent models. This study aimed to investigate whether the fixed combination with nMH improves the ocular hypotensive effect of TM and affects pupil size (PS), heart rate (HR), and mean arterial pressure (MAP) in clinically healthy dogs. Five clinically healthy dogs were administered topical saline, commercial 0.5% TM, and a 0.01% or 0.1% nMH-0.5% TM fixed combination (0.01% or 0.1% nMH-TM) twice daily in one eye for 7 days with at least a 28-day interval. The changes from baseline were calculated and were statistically analyzed for each drug. IOP was significantly reduced in both 0.01% and 0.1% nMH-TM-treated-dogs compared with saline- and TM-treated dogs. Meanwhile, 0.01% and 0.1% nMH did not exacerbate the side effects of TM. From these results, nMH improved the ocular hypotensive effect of TM without enhancing side effects. Topical nMH-TM is potentially more effective for canine ocular hypotensive eye drops than TM.

In Situ Gel Incorporating Disulfiram Nanoparticles Rescues the Retinal Dysfunction via ATP Collapse in Otsuka Long-Evans Tokushima Fatty Rats.

We attempted to design an ophthalmic in situ gel formulation incorporating disulfiram (DIS) nanoparticles (Dis-NPs/ISG) and demonstrated the therapeutic effect of Dis-NPs/ISG on retinal dysfunction in 15-month-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a rat model of diabetes. The DIS particles were crushed using a bead mill to prepare the nanoparticles, and the Dis-NPs/ISG was prepared using a combination of the DIS nanoparticles and an in situ gelling system based on methylcellulose (MC). The particle size of the Dis-NPs/ISG was 80-250 nm, and there was no detectable precipitation or aggregation for 1 month. Moreover, the Dis-NPs/ISG was gelled at 37 °C, and the drug was delivered into the retina by instillation. Only diethyldithiocarbamate (DDC) was detected in the retina (DIS was not detected) when the Dis-NPs/ISG was instilled in the right eye, and the DDC levels in the right retina were significantly higher than those in the left retina. In addition, the retinal residence time of the drug was prolonged by the application of the in situ gelling system, since the DDC levels in the retinas of rats instilled with Dis-NPs/ISG were higher than those in DIS nanoparticles without MC. Furthermore, repetitive instillation of the Dis-NPs/ISG attenuated the deterioration of electroretinograms (ERGs) in 15-month-old OLETF rats by preventing the collapse of ATP production via excessive nitric oxide and recovered the decrease in retinal function. These findings provide important information for the development of novel therapeutic approaches to diabetic retinopathy.

Decellularization of canine kidney for three-dimensional organ regeneration.

BACKGROUND AND AIM: Kidney regeneration is required for dogs with end-stage renal failure. Decellularization is one of the bioengineering techniques, which involves the removal of all tissue cells and cellular components and conservation of the extracellular matrix (ECM). Studies in rats have shown that decellularized kidney has regenerative potential; however, there are no reports on renal decellularization in dogs. Here, we showed the decellularization of the canine kidney. MATERIALS AND METHODS: The renal artery of the cadaveric canine kidney was cannulated and the whole kidney was frozen at -80°C. After completely thawing, it was perfused with physiological saline and sodium dodecyl sulfate (0.5%, 6 h) through the cannulated renal artery to achieve decellularization. To assess the efficiency of the decellularization protocol, histological and immunohistochemical analysis of decellularized kidney was performed. RESULTS: The results of hematoxylin and eosin (H and E) staining revealed that the decellularized canine kidney had no apparent cellular components. In addition, 4',6-diamidino-2-phenylindole (DAPI) staining showed no visible nuclear components within the whole decellularized kidney. Therefore, both H and E and DAPI staining showed decellularization of the canine kidney. Our decellularization protocol also preserved the basement membrane of glomerulus, shown by periodic acid methenamine silver, periodic acid-Schiff, fibronectin, and collagen type IV stain. CONCLUSION: Our decellularization protocol could eliminate cellular components and remaining native ECM structures of canine kidney. These results could promote further research into canine kidney regeneration, which may be the first small step to regenerate the canine kidney waiting for renal transplantation.

Development of Sustained-Release Ophthalmic Formulation Based on Tranilast Solid Nanoparticles.

Eye drops containing Tranilast (TL), N-(3,4-dimethoxycinnamoyl) anthramilic acid, are used as an anti-allergic conjunctivitis drug in the ophthalmic field. Traditional eye drops are very patient compliant, although the bioavailability (BA) of most eye drops is low since eye drops cannot be instilled beyond the capacity of the conjunctival sac due to its limited volume. Thus, traditional eye drops have low BA and a short duration of the drug on the ocular surface, so solutions to these problems are highly anticipated. In this study, we designed a sustained-release drug-delivery system (DDS) for TL nanoparticles. TL nanoparticles were prepared by bead mill treatment, and the gel formulations containing TL nanoparticles (TL-NPs-Gel, particle size 50 nm-100 nm) were provided by carboxypolymethylene. The crystal structure of TL with and without bead mill treatment is the same, but the TL solubility in formulations containing nanoparticles was 5.3-fold higher compared with gel formulations containing TL microparticles (TL-MPs-Gel). The photo and thermal stabilities of TL-NPs-Gel are also higher than those of dissolved TL. Moreover, when TL-NPs-Gel is applied to the upper eyelid skin (outside), the TL is released as nanoparticles, and delivered to the lacrimal fluid through the meibomian glands. In addition, the TL release profile for TL-NPs-Gel was sustained over 180 min after the treatment. These findings can be used to develop a sustained-release DDS in the ophthalmic field.

Novel Sustained-Release Drug Delivery System for Dry Eye Therapy by Rebamipide Nanoparticles.

The commercially available rebamipide ophthalmic suspension (CA-REB) was approved for clinical use in patients with dry eye; however, the residence time on the ocular surface for the traditional formulations is short, since the drug is removed from the ocular surface through the nasolacrimal duct. In this study, we designed a novel sustained-release drug delivery system (DDS) for dry eye therapy by rebamipide nanoparticles. The rebamipide solid nanoparticle-based ophthalmic formulation (REB-NPs) was prepared by a bead mill using additives (2-hydroxypropyl-ß-cyclodextrin and methylcellulose) and a gel base (carbopol). The rebamipide particles formed are ellipsoid, with a particle size in the range of 40-200 nm. The rebamipide in the REB-NPs applied to eyelids was delivered into the lacrimal fluid through the meibomian glands, and sustained drug release was observed in comparison with CA-REB. Moreover, the REB-NPs increased the mucin levels in the lacrimal fluid and healed tear film breakup levels in an N-acetylcysteine-treated rabbit model. The information about this novel DDS route and creation of a nano-formulation can be used to design further studies aimed at therapy for dry eye.

Therapeutic Potential of a Combination of Magnesium Hydroxide Nanoparticles and Sericin for Epithelial Corneal Wound Healing.

We previously found the instillation of sericin to be useful as therapy for keratopathy with or without diabetes mellitus. In this study, we investigated whether a combination of solid magnesium hydroxide nanoparticles (MHN) enhances epithelial corneal wound healing by sericin using rabbits, normal rats and type 2 diabetes mellitus rats with debrided corneal epithelium (ex vivo and in vivo studies). Ophthalmic formulations containing sericin and MHN (N-Ser) were prepared using a bead mill method. The mean particle size of the N-Ser was 110.3 nm at the time of preparation, and 148.1 nm one month later. The instillation of N-Ser had no effect on the amount of lacrimal fluid in normal rabbits (in vivo), but the MHN in N-Ser was found to expand the intercellular space in ex vivo rat corneas. In addition, the instillation of N-Ser increased the phosphorylation of Extracellular Signal-regulated Kinase (ERK)1/2, a factor involved in cell adhesion and cell proliferation in the corneal epithelium, in comparison with the instillation of sericin alone. The combination with MHN enhanced epithelial corneal wound healing by sericin in rat debrided corneal epithelium (in vivo). This study provides significant information to prepare potent drugs to cure severe keratopathy, such as diabetic keratopathy.

Preventive effects of tyrosol, a natural phenolic compound, on anterior uveitis induced by anterior chamber paracentesis in healthy beagle dogs.

We investigated the effects of tyrosol (Tyr) on anterior chamber paracentesis (ACP)-induced anterior uveitis in beagle dogs, as determined by protein and prostaglandin E2 (PGE2) concentrations in the aqueous humor (AH). Tyr at a dose of 100 or 200 mg/kg or 2.2 mg/kg of carprofen as a positive control was administered orally twice daily from 2.5 days before paracentesis. The initial ACP was performed in one eye of individual dogs and 0.5 ml AH was aspirated. The secondary AH was collected 60 min later. Pretreatment with 200 mg/kg of Tyr and carprofen significantly decreased aqueous protein and PGE2 concentrations compared to the control group. Overall, these findings suggested that Tyr was useful for the management of canine anterior uveitis.

Comparison of early posttreatment effects of two steroidal anti-inflammatory ophthalmic drugs on the ocular inflammatory response induced by paracentesis in healthy canine eyes.

OBJECTIVE: We investigated the early posttreatment effects of two steroidal anti-inflammatory ophthalmic drugs on blood-aqueous barrier (BAB) breakdown by paracentesis in dogs. ANIMAL STUDIES: We studied 21 healthy beagles with normal eyes. PROCEDURES: Controlled anterior chamber paracentesis (0.5 mL) was performed in one eye of each dog. Control group dogs (n = 7) received no medication, whereas those in the treatment groups received a topical anti-inflammatory medication (difluprednate [DFBA] ophthalmic emulsion 0.05% [n = 7] or betamethasone [BMZ] sodium phosphate ophthalmic solution 0.1% [n = 7]) at 0, 15, 30, and 45 minutes after initial paracentesis in the paracentesed eyes. Secondary aqueous humor (AH) was collected 60 minutes after initial paracentesis. Protein and prostaglandin E2 (PGE2 ) concentrations in AH were determined using the bicinchoninic acid assay and commercially available immunoassay kit, respectively. All mean values in the three groups were compared using analysis of variance followed by Tukey's post hoc test. RESULTS: Aqueous protein and PGE2 concentrations were markedly increased at 60 minutes following paracentesis. Both concentrations in the secondary AH of the DFBA group were significantly lower than those of the control group; however, treatment with BMZ had no significant effects. CONCLUSIONS: Early postparacentesis treatment with DFBA was more effective than that with BMZ for reducing aqueous protein and PGE2 contents in dogs with paracentesis-induced BAB breakdown. DFBA may be an appropriate treatment during the early stage of anterior uveitis caused by intraocular surgery in dogs.

Enhancement in Corneal Permeability of Dissolved Carteolol by Its Combination with Magnesium Hydroxide Nanoparticles.

We prepared magnesium hydroxide (MH) nanoparticles, and investigated their effect when combined with dissolved carteolol on the bioavailability and intraocular pressure (IOP)-reducing effect of carteolol. The carteolol was solved in saline containing additives (0.5% methylcellulose, 0.001% benzalkonium chloride, 0.5% mannitol; CRT-solution). MH nanoparticles were prepared by a bead mill method with additives. Then carteolol/MH microparticle and carteolol/MH nanoparticle fixed combinations (mCMFC and nCMFC) were prepared by mixing the CRT-solution and MH particles. The transcorneal penetration and IOP-reducing effect of carteolol was evaluated in rabbits. The mean particle size of mCMFC was 7.2 µm, and the particle size was reduced to 73.5-113.5 nm by the bead mill treatment. The MH particles in nCMFC remained in the nano size range for 8 days after preparation, and the amounts of lacrimal fluid and corneal damage were unchanged by repetitive instillation of nCMFC (twice a day for 4 weeks). The transcorneal penetration of carteolol was enhanced by the combination with MH nanoparticles, and the IOP-reducing effect of nCMFC was significantly higher than that of CRT-solution or mCMFC. In conclusion, we designed nCMFC, and showed that the high levels of dissolved carteolol can be delivered into the aqueous humor by the instillation of nCMFC. Combination with MH nanoparticles may achieve an enhancement of corneal penetration for water-soluble drugs. These findings provide significant information that can be used to design further studies aimed at developing anti-glaucoma eye drugs.

Co-instillation of nano-solid magnesium hydroxide enhances corneal permeability of dissolved timolol.

We prepared magnesium hydroxide (MH) nanoparticles by a bead mill method, and investigated whether the co-instillation of MH nanoparticles improves the low transcorneal penetration of water-soluble drugs, such as the anti-glaucoma eye drug timolol maleate (TM). MH particle size was decreased by the bead mill treatment to a mean particle size of 71 nm. In addition, the MH nanoparticles were highly stable. Next, we demonstrated the effect of MH nanoparticles on the corneal surface. MH shows only slight solubility in lacrimal fluid, and the instillation of MH nanoparticles for 14 days did not affect the behavior (balance of secretion and excretion) of the lacrimal fluid in rabbit corneas. Moreover, there was no observable corneal toxicity of MH nanoparticles, and treatment with MH nanoparticles enhanced the intercellular space ratio in the eyes of rats. MH alone did not permeate into the cornea; however, the co-instillation of MH nanoparticles and dissolved TM (nMTFC) enhanced the corneal penetration of TM. In addition, the intraocular pressure (IOP)-reducing effect of nMTFC was significantly higher than those of the TM solution or the co-instillation of MH microparticles and TM. In conclusion, we found that MH nanoparticles enhance the corneal penetration of dissolved TM with no observable corneal stimulation or obstruction of the nasolacrimal duct by the MH nanoparticles. It is possible that the co-instillation of MH nanoparticles may provide a useful way to improve the bioavailability of water-soluble drugs in the ophthalmic field. These findings provide significant information that can be used to design further studies aimed at developing anti-glaucoma eye drugs.

The Aldosterone Receptor Antagonist Eplerenone Inhibits Isoproterenol-Induced Collagen-I and 11ß-HSD1 Expression in Rat Cardiac Fibroblasts and the Left Ventricle.

ß-Adrenergic receptor (ß-AR)-induction of collagen-I synthesis is partially mediated by the cardiac mineralocorticoid receptor (MR) system. However, it remains unclear whether the selective MR antagonist, eplerenone, inhibits collagen-I synthesis induced by ß-AR stimulation. We investigated the effects of eplerenone on the responses to a non-selective ß-AR agonist, isoproterenol, which induced collagen-I synthesis in primary cardiac fibroblasts (CFs) and the left ventricle. mRNAs encoding the MR and 11ß-hydroxysteroid dehydrogenase type I (11ß-HSD1) were evident in the left ventricle and primary CFs. mRNAs encoding the CYP family 11 subfamily B member 2 (CYP11-B2) were not detected, even after isoproterenol treatment. In vivo, isoproterenol induced collagenous fiber accumulation in the left ventricle. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), 11ß-HSD1 levels, and mRNA/protein levels of collagen-I increased upon exposure to isoproterenol, but these increases were inhibited by eplerenone co-treatment. In primary CFs, isoproterenol increased the phosphorylation of ERK1/2 and the expression levels of both 11ß-HSD1 and collagen-I; these isoproterenol-attributable effects were inhibited by co-treatment with eplerenone and PD98059, a specific inhibitor of mitogen-activated protein kinase/ERK kinase activity. The results suggest that 11ß-HSD1 but not CYP11-B2 is expressed in primary CFs. Eplerenone inhibited isoproterenol-induced ERK1/2 phosphorylation and expression of 11ß-HSD1 and collagen-I in primary CFs, as well as the progression of cardiac fibrosis in the left ventricle. Therefore, eplerenone inhibited the isoproterenol-induced increases in 11ß-HSD1 and collagen-I expression in primary CFs, and progression of cardiac fibrosis in the left ventricle.

Repeated phlebotomies decrease body iron storage in adult dogs.

The aim of the present study was to determine changes in body iron storage in adult dogs following phlebotomy. We performed repeated phlebotomies by removing 1% body weight (approximately 10% of the total blood volume) weekly for a total of 12 times using adult beagle dogs without an iron-restricted diet. After treatment, stored iron was decreased, as demonstrated by gradual reductions in serum ferritin levels and hepatic iron contents. Anemia and abnormalities in blood chemistry analysis were not observed; therefore, this method was considered safe and useful for control of stored iron levels in adult dogs.