Novel Herbal Therapeutic YH23537 Improves Clinical Parameters in Ligature-Induced Periodontal Disease Model in Beagle Dogs.

Currently, available medicine does not satisfy the clinical unmet needs of periodontal disease. Therefore, novel drugs with improved efficacy profiles are needed. We previously demonstrated that YH14642, water extracts of Notoginseng Radix and Rehmanniae Radix Preparata, improved probing depths in double-blind phase II clinical trial. However, it still has hurdles for commercialization due to the low efficiency of active compound extraction. To resolve this issue, we developed YH23537 through process optimization to extract active compounds efficiently while still achieving the chemical profile of YH14642. In this study, we investigated the therapeutic effects of YH23537 compared with YH14642 using a canine model of ligature-induced periodontitis. Human gingival fibroblast (hGF) cells were treated with various concentrations of YH23537 or YH14642 with lipopolysaccharide (LPS) for 24 hr. IL-6 and IL-8 levels in the conditioned media were determined using Luminex. Sixteen 3-year-old male beagle dogs had their teeth scaled and polished using a piezo-type ultrasonic scaler under general anesthesia and brushed once daily for the following 2 weeks. Two weeks after the scaling procedure, the left upper second premolar (PM2), third premolar (PM3), and fourth premolar (PM4) as well as the left lower PM3, PM4, and first molar (M1) were ligated with silk-wire twisted ligatures. The dogs were fed with soft moistened food to induce periodontitis for 8 weeks, and the ligatures were then removed. YH23537 and YH14642 were administered for 4 weeks, and clinical periodontal parameters such as plaque index (PI), gingival index (GI), probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BoP) were determined before and 1, 2, 3, and 4 weeks after treatment. YH23537 inhibited IL-6 and IL-8 secretion in a dose-dependent manner in hGF cells stimulated with LPS. The IC50 values for YH23537 were 43 and 54 µg/ml for IL-6 and IL-8, respectively, while the values for YH14642 were 104 and 117 µg/ml, respectively. In the animal study, clinical parameters including GI, PD, CAL, and BoP were significantly increased after 8 weeks of ligature-induced periodontitis. The YH23537 300 and YH23537 900 mg groups had significant improvements in CAL from 1 to 4 weeks after treatment in comparison to the placebo group. GR values in the YH23537 900 mg group were decreased throughout the treatment period. GI values were also reduced significantly after 4-week treatment with 300 and 900 mg of YH23537. YH23537 at 300 mg doses showed comparable efficacy for CAL and GR with 1,000 mg of YH14642. YH23537 showed therapeutic efficacy against periodontitis in dogs, mediated by anti-inflammatory effects. These findings indicate that YH23537 has the potential for further development as a new drug for patients suffering from periodontal disease.

Ultrasound biomicroscopic study of the effects of topical latanoprost on the anterior segment and ciliary body thickness in dogs.

OBJECTIVE: The goal of this study was to evaluate the effects of topical latanoprost on the anterior segment and ciliary body using ultrasound biomicroscopy (UBM) in dogs. ANIMALS STUDIED: This study included six eyes of six clinically normal beagles. PROCEDURES: UBM scans were performed on six sedated dogs before and 2 h after topical latanoprost instillation. From the next day on, latanoprost was topically applied twice daily for 7 days. After 1 week of instillation, the UBM scans were repeated. The ciliary body thickness (CBT) and the anterior segment parameters, including the iridocorneal angle (ICA), the width of the ciliary cleft (CC) entry, the length of the CC, and the width of the mid-CC, were measured. RESULTS: The topical latanoprost decreased the ICA and CC entry width and increased the mid-CC width without any significant alterations in the CC length. There were time-dependent alterations in the CBT: a reduction in the CBT after 2 h of instillation and rebound thickening after 1 week of instillation. CONCLUSIONS: The topical latanoprost widened the ciliary cleft despite the narrowing of the ICA and CC entry. Time-dependent alterations in the CBT were demonstrated by the UBM and might be a reflection of the mechanism of the uveoscleral outflow enhancement induced by the topical latanoprost.

Effect of onion extract on corneal haze suppression after air assisted lamellar keratectomy.

This study evaluated the effect of onion extract on corneal haze suppression after applying the air assisted lamellar keratectomy. The air assisted lamellar keratectomy was performed on 24 canine eyes. They were treated with an artificial tear (group C), prednisolone acetate (group P), onion extract (group O) and TGF-ß1 (group T) three times per day from 7 to 28 days after the surgery. Corneal haze occurred on the all eyes and was observed beginning 7 days after the surgery. The haze was significantly decreased in groups P and O from day 14 compared with the group C using the clinical (group P; P=0.021, group O; P=0.037) and objective evaluation method (group P; P=0.021, group O; P=0.039). In contrast, it was significantly increased in group T from day 14 compared with group C based on the clinical (P=0.002) and objective evaluation method (P<0.001). Subsequently, these eyes were enucleated after euthanasia, and immunohistochemistry with α-SMA antibodies was done. The total green intensity for α-SMA was significantly more expressed in group T and significantly less expressed in groups P and O than in group C. Onion extract could have potential as a therapeutic in preventing corneal haze development by suppressing the differentiation of fibroblasts into myofibroblasts.

Air assisted lamellar keratectomy for the corneal haze model.

To standardize the corneal haze model in the resection depth and size for efficient corneal haze development, air assisted lamellar keratectomy was performed. The ex vivo porcine corneas were categorized into four groups depending on the trephined depth: 250 µm (G1), 375 µm (G2), 500 µm(G3) and 750 µm (G4). The stroma was equally ablated at the five measurement sites in all groups. Significant differences were observed between the trephined corneal depths for resection and ablated corneal thickness in G1 (p < 0.001). No significant differences were observed between the trephined corneal depth for resection and the ablated corneal thickness in G2, G3, and G4. The resection percentage was similar in all groups after microscopic imaging of corneal sections. Air assisted lamellar keratectomy (AK) and conventional keratectomy (CK) method were applied to six beagles, after which development of corneal haze was evaluated weekly until postoperative day 28. The occurrence of corneal haze in the AK group was significantly higher than that in the CK group beginning 14 days after surgery. Alpha-smooth muscle actin expression was significantly higher in the AK group (p < 0.001) than the CK group. Air assisted lamellar keratectomy was used to achieve the desired corneal thickness after resection and produce sufficient corneal haze.

Evaluation of fluid leakage into the canine vitreous humor during phacoemulsification using contrast-enhanced magnetic resonance imaging.

OBJECTIVE: To evaluate the effects of phacoemulsification with different fluidic parameters on the intraocular tissues using contrast-enhanced magnetic resonance imaging (CE-MRI). PROCEDURES: Phacoemulsification with intraocular lens (IOL) implantation was performed on 10 pairs of enucleated canine eyes. Irrigation fluid containing diluted MRI contrast agent was used, with the bottle height/the vacuum pressure set at 50 cm/80 mmHg for one eye (Group L) and at 120 cm/150 mmHg for the opposite eye (Group H). CE-MRI was carried out immediately after phacoemulsification to evaluate the presence of anterior hyaloid membrane detachment (AHD) and the leakage volume-to-vitreous volume ratio (LV/VV%). The ultrasound time, the volume of irrigation solution used, and the total irrigation time were recorded. RESULTS: AHD was seen in seven of the 10 eyes in Group L and in nine of the 10 eyes in Group H. Fluid leakage into the vitreous humor (LV/VV%) was significantly greater in Group H than in Group L (P < 0.01). The LV/VV% was also correlated with the total irrigation time in both groups (P < 0.05). The volume of irrigation solution used in Group H was significantly greater than that used in Group L (P < 0.001). There was no statistically significant difference in the ultrasound time between the two groups. CONCLUSIONS: Low fluidic parameters led to decreased fluid passage through the zonules, reducing fluid passage into the vitreous. These may also lead to reduced surgical stress on the intraocular tissues by decreasing the amount of irrigation solution used during phacoemulsification in the dog.

Determining the age of cats by pulp cavity/tooth width ratio using dental radiography.

The purpose of this study was to evaluate the effect of age on the ratio of pulp cavity/tooth width (P/T ratio) in healthy cats. The dental radiographs of 32 cats (16 males and 16 females) were generated with a digital dental X-ray unit with the animals under general anesthesia. Standardized measurement of the canine teeth was performed by drawing a line on the radiograph perpendicular to the cemento- enamel junction (CEJ) of the tooth. There was an inversely proportional correlation between chronological age and the P/T ratio. Moreover, a strong Pearson squared correlation (γ(2) = 0.92) was identified by the curved regression model. No significant differences in the P/T ratio based on gender or breed were found. These results suggest that determination of age by P/T ratio could be clinically useful for estimating the chronological age of cats.

Effects of peribulbar anesthesia (sub-Tenon injection of a local anesthetic) on akinesia of extraocular muscles, mydriasis, and intraoperative and postoperative analgesia in dogs undergoing phacoemulsification.

OBJECTIVE: To evaluate the effects of peribulbar anesthesia (sub-Tenon injection of lidocaine hydrochloride) on akinesia of extraocular muscles, mydriasis, and intraoperative and postoperative analgesia in dogs undergoing phacoemulsification. ANIMALS: 14 Beagles with ophthalmically normal eyes. PROCEDURES: A blinded randomized controlled trial was performed. Dogs were anesthetized and assigned to 2 treatments: concurrent sub-Tenon injection of 2% lidocaine hydrochloride solution (2 mL) and IV injection of saline (0.9% NaCl) solution (0.02 mL/kg; lidocaine group [n = 7]) or concurrent sub-Tenon injection of saline solution (2 mL) and IV injection of 0.2 mg of atracurium/kg (0.02 mL/kg; control group [7]). Pupils were dilated by topical application of a combined tropicamide and phenylephrine ophthalmic solution. Ten minutes after the injections, pupil diameter was measured and phacoemulsification was performed. End-tidal isoflurane concentration was used to evaluate intraoperative pain. Subjective pain scores were recorded during the postoperative period. RESULTS: Akinesia was induced and maintained throughout the surgery in all eyes. Mean ± SD pupil diameter was significantly greater in the lidocaine group (13.7 ± 0.7 mm) than in the control group (12.2 ± 0.8 mm). Isoflurane requirements were significantly lower in the lidocaine group than the control group. However, postoperative pain scores were not significantly different between the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Sub-Tenon injection of lidocaine was an effective method for inducing akinesia of extraocular muscles, mydriasis, and intraoperative analgesia for phacoemulsification in dogs. Therefore, this could be another option for surgical field exposure and pain management during phacoemulsification in dogs.

Comparison of systemic atracurium, retrobulbar lidocaine, and sub-Tenon's lidocaine injections in akinesia and mydriasis in dogs.

OBJECTIVE: To compare the effect of sub-Tenon's lidocaine injections (ST) on akinesia and mydriasis to those of systemic atracurium (AT) and retrobulbar lidocaine injections in dogs. ANIMAL STUDIED: Ten healthy beagle dogs without apparent ocular disease. PROCEDURES: Three treatments were performed on 10 beagle dogs with a minimum 7-day washout period: intravenous injection of AT (0.2 mg/kg, AT group); retrobulbar (RB) injection of 2% lidocaine (2.0 mL, RB group) in one eye; and sub-Tenon's injection of 2% lidocaine (2.0 mL, ST group) in the opposite eye. When the akinesia was not obtained within 10 min, an additional 1 mL of lidocaine was administered in the RB and the ST groups. RESULTS: Onset of akinesia in the AT (1.5 ± 0.9 min) and the ST (3.8 ± 5.8 min) groups was significantly shorter than that in the RB group (9.0 ± 6.5 min). Duration of akinesia in the ST group (116.2 ± 32.8 min) was longer compared to the AT (60.6 ± 23.6 min) and the RB (89.0 ± 52.8 min) groups, even though there was only a significant difference between the AT and the ST groups. Mydriasis was achieved in five eyes in the RB group and nine eyes in the ST group. There was no significant difference in onset (3.6 ± 3.1 and 2.9 ± 2.3 min, respectively) or duration (91.4 ± 31.9 and 102.1 ± 35.8 min, respectively) of mydriasis between the groups. CONCLUSIONS: Sub-Tenon's lidocaine injections provide excellent akinesia and mydriasis compared to systemic AT and retrobulbar lidocaine injections. Therefore, sub-Tenon's anesthesia could be an alternative to the systemic administration of neuromuscular blockers and retrobulbar anesthesia for ophthalmic surgery in dogs.

Experimental determination of a subantimicrobial dosage of doxycycline hyclate for treatment of periodontitis in Beagles.

OBJECTIVE: To identify a subantimicrobial dose of doxycycline hyclate (SDD) and for the treatment of periodontitis in dogs. ANIMALS: 20 healthy Beagles for measurement of serum doxycycline concentration and 15 Beagles with periodontitis for evaluation of the efficacy of the SDD. PROCEDURES: 5 dogs each received doxycycline hyclate PO at a dose of 1, 2, 3, or 5 mg/kg. Blood samples were collected before and after administration, and serum concentrations of doxycycline were measured via high-performance liquid chromatography. Mean serum doxycycline concentrations were calculated, and SDDs were identified. In a separate trial, the identified SDDs (1 or 2 mg/kg) were administered PO once a day for 1 month to dogs with periodontitis (n = 5/group) and a control group (5) was fed vehicle only during the same period. Degree of gingival attachment and bleeding on probing (present or absent) were recorded. Gingival samples were collected before and after the 1-month period from the same anatomic sites. Degree of matrix metalloproteinase inhibition in gingival samples was determined via gelatin zymography and compared among treatment groups. RESULTS: Mean serum doxycycline concentrations in healthy dogs that received 1 or 2 mg of doxycycline/kg were consistently significantly lower than the minimal inhibitory doxycycline concentration for treatment of periodontitis throughout the 24-hour posttreatment period. Zymographic intensities were lower in dogs given 1 and 2 mg/kg than in the control dogs, and the degree of gingival attachment and bleeding significantly improved in dogs given 2 mg/kg, compared with in the control dogs and dogs given 1 mg of doxycycline/kg. CONCLUSIONS AND CLINICAL RELEVANCE: A doxycycline dosage of 2 mg/kg daily appeared to be an appropriate subantimicrobial regimen for dogs with periodontitis. Furthermore, this dosage may be suitable for long-term treatment of gelatinolytic inflammatory diseases such as periodontitis in this species.

Accuracy of intraocular pressure measurements in dogs using two different tonometers and plano therapeutic soft contact lenses.

OBJECTIVE: To compare and evaluate the accuracy of intraocular pressure (IOP) measured through a therapeutic contact lens, using applanation (TonoPen XL(®)) and rebound (TonoVet(®)) tonometers in enucleated dog eyes. ANIMALS STUDIED: A total of 30 enucleated eyes from 15 beagle dogs. PROCEDURES: To measure accurate IOP, the anterior chamber of each enucleated eye was cannulated with two 26-gauge needles and two polyethylene tubes were connected vertically to an adjustable reservoir bag of normal saline and a pressure transducer. IOP was measured by the TonoPen XL(®) followed by the TonoVet(®) without a contact lens. After a contact lens was applied to the cornea, IOP was re-measured in the same order. Three consecutive IOP measurements were performed using both tonometers. RESULTS: Without the contact lens, the IOP values obtained by both tonometers correlated well according to the regression analysis (TonoVet(®): γ(2) = 0.98, TonoPen XL(®): γ(2) = 0.97, P < 0.001). The TonoPen XL(®) consistently underestimated values as transducer IOP increased; however, IOP values measured with the TonoPen XL(®) were in close agreement and were less variable than those determined with the TonoVet(®) when a contact lens was applied to the cornea. Bland-Altman analysis was used to determine the lower and upper limits of agreement (TonoVet(®): -29.7 and +21.1 mmHg, TonoPen XL(®): -3.9 and +3.6 mmHg) between the two devices. CONCLUSIONS: This study suggests that the TonoPen XL(®) is a useful tonometer for dogs wearing therapeutic contact lenses, and importantly, contact lenses would not need to be removed prior to IOP measurement.