Quantitative orbital tightening for pain assessment using machine learning with DeepLabCut.

Pain assessment in animal models is essential for understanding mechanisms underlying pathological pain and developing effective pain medicine. The grimace scale (GS), facial expression features in pain such as orbital tightening (OT), is a valuable measure for assessing pain in animal models. However, the classical grimace scale for pain assessment is labor-intensive, subject to subjectivity and inconsistency, and is not a quantitative measure. In the present study, we utilized machine learning with DeepLabCut to annotate the superior and inferior eyelid margins and the medial and lateral canthus of the eyes in animals' video images. Based on the annotation, we quantified the eyelid distance and palpebral fissure width of the animals' eyes so that the degree of OT in animals with pain could be measured and described quantitatively. We established criteria for the inclusion and exclusion of the annotated images for quantifying OT, and validated our quantitative grimace scale (qGS) in the mice with pain caused by capsaicin injections in the orofacial or hindpaw regions, the Nav1.8-ChR2 mice following orofacial noxious stimulation with laser light, and the oxaliplatin-treated mice following tactile stimulation with a von Frey filament. We showed that both the eyelid distance and the palpebral fissure width were shortened significantly in the animals in pain compared to the control animals without nociceptive stimulation. Collectively, the present study has established a quantitative orbital tightening for pain assessment in mice using DeepLabCut, providing a new tool for pain assessment in preclinical studies with mice.

Gender inclusivity of ophthalmology journal submission guidelines and associated research metrics.

PURPOSE: This cross-sectional study evaluated the prevalence of inclusive author submission guidelines across ophthalmology journals. METHODS: Journals were identified from the 2021 Journal Citations Report (Clarivate Analytics). Independent reviewers rated each author submission guideline as "inclusive" for satisfying at-least one of six criteria: i) included examples of gender inclusive language; ii) recommended the use of gender-inclusive language; iii) distinguished between sex and gender; iv) provided educational resources on gender-inclusive language; v) provided a policy permitting name changes (e.g., in case of gender and name transition); and/or vi) provided a statement of commitment to inclusivity. The primary objective was to investigate the proportion of journals with "gender-inclusive" author submission guidelines and the elements of the gender-inclusive content within these guidelines. A secondary objective was to review the association between "gender-inclusivity" in author submission guidelines with publisher, origin country, and journal/source/influence metrics (Clarivate Analytics). RESULTS: Across 94 journals, 29.8% journals were rated as inclusive. Inclusive journals had significantly higher relative impact factor, citations, and article influence scores compared to non-inclusive journals. Of the 29.8% of inclusive journals, the three most common domains were inclusion of an inclusivity statement (71.4% of inclusive journals), distinguishing between sex and gender (67.9%), and provision of additional educational resources on gender reporting for authors (60.7%). CONCLUSION: A minority of ophthalmology journals have gender-inclusive author submission guidelines. Ophthalmology journals should update their submission guidelines to advance gender equity of both authors and study participants and promote the inclusion of gender-diverse communities.

Assessment of orofacial nociceptive behaviors of mice with the sheltering tube method: Oxaliplatin-induced mechanical and cold allodynia in orofacial regions.

Preclinical studies on pathological pain rely on the von Frey test to examine changes in mechanical thresholds and the acetone spray test to determine alterations in cold sensitivity in rodents. These tests are typically conducted on rodent hindpaws, where animals with pathological pain show reliable nocifensive responses to von Frey filaments and acetone drops applied to the hindpaws. Pathological pain in orofacial regions is also an important clinical problem and has been investigated with rodents. However, performing the von Frey and acetone spray tests in the orofacial region has been challenging, largely due to the high mobility of the head of testing animals. To solve this problem, we implemented a sheltering tube method to assess orofacial nociception in mice. In experiments, mice were sheltered in elevated tubes, where they were well accommodated because the tubes provided safe shelters for mice. Examiners could reliably apply mechanical stimuli with von Frey filament, cold stimuli with acetone spray, and light stimuli with a laser beam to the orofacial regions. We validated this method in Nav1.8-ChR2 mice treated with oxaliplatin that induced peripheral neuropathy. Using the von Frey test, orofacial response frequencies and nociceptive response scores were significantly increased in Nav1.8-ChR2 mice treated with oxaliplatin. In the acetone spray test, the duration of orofacial responses was significantly prolonged in oxaliplatin-treated mice. The response frequencies to laser light stimulation were significantly increased in Nav1.8-ChR2 mice treated with oxaliplatin. Our sheltering tube method allows us to reliably perform the von Frey, acetone spray, and optogenetic tests in orofacial regions to investigate orofacial pain.

Distinct local and global functions of mouse Aß low-threshold mechanoreceptors in mechanical nociception.

The roles of Aß low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of SplitCre labeled mouse Aß-LTMRs in this regard. Genetic ablation of SplitCre-Aß-LTMRs increased mechanical nociception but not thermosensation in both acute and chronic inflammatory pain conditions, indicating a modality-specific role in gating mechanical nociception. Local optogenetic activation of SplitCre-Aß-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a model, in which Aß-LTMRs play distinctive local and global roles in transmitting or alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a strategy of global activation plus local inhibition of Aß-LTMRs for treating mechanical hyperalgesia.

Trends and Clinical Outcomes of Fungal Keratitis in Canada: A 20-year Retrospective Multicentre Study.

PURPOSE: An increase in fungal and particularly filamentous keratitis has been observed in many geographic areas, mostly in contact lens wearers. This study seeks to characterize long-term trends in fungal keratitis in a continental climate area to provide guidance for diagnosis and treatment. DESIGN: Retrospective multicentric case series. METHODS: Cases of microbiology-confirmed fungal keratitis from 2003 to 2022 presenting to tertiary care centers across Canada were included. Charts were reviewed for patient demographics, risk factors, visual acuity, and treatments undertaken. RESULTS: A total of 138 patients were identified: 75 had yeast keratitis while 63 had filamentous keratitis. Patients with yeast keratitis had more ocular surface disease (79% vs 28%) while patients with filamentous keratitis wore more refractive contact lenses (78% vs 19%). Candida species accounted for 96% of all yeast identified, while Aspergillus (32%) and Fusarium (26%) were the most common filamentous fungi species. The mean duration of treatment was 81 ± 96 days. Patients with yeast keratitis did not have significantly improved visual acuity with medical treatment (1.8 ± 1 LogMAR to 1.9 ± 1.5 LogMAR, P = .9980), in contrast to patients with filamentous keratitis (1.4 ± 1.2 LogMAR to 1.1 ± 1.3 LogMAR, P = .0093). CONCLUSIONS: Fungal keratitis is increasing in incidence, with contact lenses emerging as one of the leading risk factors. Significant differences in the risk factors and visual outcomes exist between yeast keratitis and filamentous keratitis which may guide diagnosis and treatment.

Effects of inflammation on the properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

We recently used Nav1.8-ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8-ChR2 mice received a subcutaneous injection of saline or Complete Freund's Adjuvant (CFA) in the hindpaws. Using the hind paw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hind paw inflammation lowered the mechanical threshold of many Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8-ChR2-negative Aß-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8-ChR2-positive Aß-fiber mechanoreceptors but occurred in Nav1.8-ChR2-negative Aß-fiber mechanoreceptors with a lower mechanical threshold in the saline goup, and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors and Nav1.8-ChR2-positive C-fiber mechanoreceptors following hind paw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aß-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.

ASICs mediate fast excitatory synaptic transmission for tactile discrimination.

Tactile discrimination, the ability to differentiate objects' physical properties such as texture, shape, and edges, is essential for environmental exploration, social interaction, and early childhood development. This ability heavily relies on Merkel cell-neurite complexes (MNCs), the tactile end-organs enriched in the fingertips of humans and the whisker hair follicles of non-primate mammals. Although recent studies have advanced our knowledge on mechanical transduction in MNCs, it remains unknown how tactile signals are encoded at MNCs. Here, using rodent whisker hair follicles, we show that tactile signals are encoded at MNCs as fast excitatory synaptic transmission. This synaptic transmission is mediated by acid-sensing ion channels (ASICs) located on the neurites of MNCs, with protons as the principal transmitters. Pharmacological inhibition or genetic deletion of ASICs diminishes the tactile encoding at MNCs and impairs tactile discrimination in animals. Together, ASICs are required for tactile encoding at MNCs to enable tactile discrimination in mammals.

Distinct Local and Global Functions of Aß Low-Threshold Mechanoreceptors in Mechanical Pain Transmission.

The roles of Aß low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of SplitCre labeled Aß-LTMRs in this regard. Genetic ablation of SplitCre-Aß-LTMRs increased mechanical pain but not thermosensation in both acute and chronic inflammatory pain conditions, indicating their modality-specific role in gating mechanical pain transmission. Local optogenetic activation of SplitCre-Aß-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a new model, in which Aß-LTMRs play distinctive local and global roles in transmitting and alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a new strategy of global activation plus local inhibition of Aß-LTMRs for treating mechanical hyperalgesia.

Distinct Local and Global Functions of Aß Low-Threshold Mechanoreceptors in Mechanical Pain Transmission.

The roles of Aß low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of Split Cre labeled Aß-LTMRs in this regard. Genetic ablation of Split Cre -Aß-LTMRs increased mechanical pain but not thermosensation in both acute and chronic inflammatory pain conditions, indicating their modality-specific role in gating mechanical pain transmission. Local optogenetic activation of Split Cre -Aß-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a new model, in which Aß-LTMRs play distinctive local and global roles in transmitting and alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a new strategy of global activation plus local inhibition of Aß-LTMRs for treating mechanical hyperalgesia.

Visual Outcomes of Cataract Surgery in Patients With Keratoconus Using Toric and Non-toric Lenses.

PURPOSE: To compare the accuracy and outcomes of different intraocular lens (IOL) power calculation formulas in eyes with keratoconus undergoing cataract surgery with toric and non-toric IOLs. METHODS: This was a consecutive retrospective case series study including patients from the Cornea Service at the Department of Ophthalmology and Visual Sciences at the University of British Columbia, Vancouver, Canada, from 2000 to 2020. Keratoconus was diagnosed based on corneal topography and clinician opinion. Patients who underwent topography-guided photorefractive keratectomy, intracorneal ring segments implantation, or corneal transplant were excluded. The manifest spherical equivalent, prediction errors, and median absolute errors were calculated. Descriptive statistics were expressed as mean ± standard deviation. RESULTS: There were 160 eyes from 101 patients; 136 eyes received non-toric lenses and 24 eyes received toric lenses. Most patients had mild disease (< 48.00 diopters [D]) when stratified by steep keratometry values. Patients with severe disease (> 53.00 D) were significantly more hyperopic following surgery (P < .05). The Barrett Universal II (0.26 D, inter-quartile range [IQR] = 0.4), Holladay 2 (0.31, IQR = 1.2), and SRK/T (0.42, IQR = 0.86) formulas had the lowest median absolute error. The postoperative prediction error following toric lens insertion was not significantly different than following non-toric lens insertion, and the mean absolute astigmatism was significantly reduced with toric lenses. CONCLUSIONS: The Barrett Universal II, Holladay 2, and SRK/T were the most accurate IOL power calculation formulas in patients with keratoconus undergoing cataract surgery. Hyperopic surprise was increased in severe keratoconus. Toric IOLs may be considered in patients with mild keratoconus. [J Refract Surg. 2023;39(5):319-325.].

Superior limbic keratoconjunctivitis: Update on pathophysiology and management.

Superior limbic keratoconjunctivitis (SLK) is an under-recognized condition characterized by a final common pathologic presentation of superior conjunctival and limbal inflammation and staining. Existing literature attributes both microtrauma and local inflammation, frequently in the setting of tear film insufficiency, as the underlying mechanisms that lead to a self-perpetuating pathologic process dependent in on inflammatory cells and signaling. Effective treatments act by targeting inflammation and by mitigating mechanical stressors. This critical review discusses the latest in our understanding of the pathophysiology of SLK and how it guides our treatment strategies.

Properties of Nav1.8ChR2-positive and Nav1.8ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice.

Nav1.8-positive afferent fibers are mostly nociceptors playing a role in mediating thermal and mechanical pain, but mechanoreceptors within these afferents have not been fully investigated. In this study, we generated mice expressing channel rhodopsin 2 (ChR2) in Nav1.8-positive afferents (Nav1.8ChR2), which showed avoidance responses to mechanical stimulation and nocifensive responses to blue light stimulation applied to hindpaws. Using ex vivo hindpaw skin-tibial nerve preparations made from these mice, we characterized properties of mechanoreceptors on Nav1.8ChR2-positive and Nav1.8ChR2-negative afferent fibers that innervate the hindpaw glabrous skin. Of all Aß-fiber mechanoreceptors, small portion was Nav1.8ChR2-positive. Of all Aδ-fiber mechanoreceptors, more than half was Nav1.8ChR2-positive. Of all C-fiber mechanoreceptors, almost all were Nav1.8ChR2-positive. Most Nav1.8ChR2-positive Aß-, Aδ-, and C-fiber mechanoreceptors displayed slowly adapting (SA) impulses in response to sustained mechanical stimulation, and their mechanical thresholds were high in the range of high threshold mechanoreceptors (HTMRs). In contrast, sustained mechanical stimulation applied to Nav1.8ChR2-negative Aß- and Aδ-fiber mechanoreceptors evoked both SA and rapidly adapting (RA) impulses, and their mechanical thresholds were in the range of low threshold mechanoreceptors (LTMRs). Our results provide direct evidence that in the mouse glabrous skin, most Nav1.8ChR2-negative Aß-, Aδ-fiber mechanoreceptors are LTMRs involving in the sense of touch, whereas Nav1.8ChR2-positive Aß-, Aδ-, and C-fiber mechanoreceptors are mainly HTMRs involving in mechanical pain.

Function of KCNQ2 channels at nodes of Ranvier of lumbar spinal ventral nerves of rats.

Previous immunohistochemical studies have shown the expression of KCNQ2 channels at nodes of Ranvier (NRs) of myelinated nerves. However, functions of these channels at NRs remain elusive. In the present study, we addressed this issue by directly applying whole-cell patch-clamp recordings at NRs of rat lumbar spinal ventral nerves in ex vivo preparations. We show that depolarizing voltages evoke large non-inactivating outward currents at NRs, which are partially inhibited by KCNQ channel blocker linopirdine and potentiated by KCNQ channel activator retigabine. Furthermore, linopirdine significantly alters intrinsic electrophysiological properties of NRs to depolarize resting membrane potential, increase input resistance, prolong AP width, reduce AP threshold, and decrease AP amplitude. On the other hand, retigabine significantly decreases input resistance and increases AP rheobase at NRs. Moreover, linopirdine increases excitability at NRs by converting single AP firing into multiple AP firing at many NRs. Saltatory conduction velocity is significantly reduced by retigabine, and AP success rate at high stimulation frequency is significantly increased by linopirdine. Collectively, KCNQ2 channels play a significant role in regulating intrinsic electrophysiological properties and saltatory conduction at NRs of motor nerve fibers of rats. These findings may provide insights into how the loss-of-function mutation in KCNQ2 channels can lead to neuromuscular disorders in human patients.

Branched Chain Amino Acid Supplementation to a Hypocaloric Diet Does Not Affect Resting Metabolic Rate but Increases Postprandial Fat Oxidation Response in Overweight and Obese Adults after Weight Loss Intervention.

BACKGROUND: Branched chain amino acids (BCAA) supplementation is reported to aid in lean mass preservation, which may in turn minimize the reduction in resting metabolic rate (RMR) during weight loss. Our study aimed to examine the effect of BCAA supplementation to a hypocaloric diet on RMR and substrate utilization during a weight loss intervention. METHODS: A total of 111 Chinese subjects comprising 55 males and 56 females aged 21 to 45 years old with BMI between 25 and 36 kg/m2 were randomized into three hypocaloric diet groups: (1) standard-protein (14%) with placebo (CT), (2) standard-protein with BCAA, and (3) high-protein (27%) with placebo. Indirect calorimetry was used to measure RMR, carbohydrate, and fat oxidation before and after 16 weeks of dietary intervention. RESULTS: RMR was reduced from 1600 ± 270 kcal/day to 1500 ± 264 kcal/day (p < 0.0005) after weight loss, but no significant differences in the change of RMR, respiratory quotient, and percentage of fat and carbohydrate oxidation were observed among the three diet groups. Subjects with BCAA supplementation had an increased postprandial fat (p = 0.021) and decreased postprandial carbohydrate (p = 0.044) oxidation responses compared to the CT group after dietary intervention. CONCLUSIONS: BCAA-supplemented standard-protein diet did not significantly attenuate reduction of RMR compared to standard-protein and high-protein diets. However, the postprandial fat oxidation response increased after BCAA-supplemented weight loss intervention.

Effects of GABAB receptor activation on excitability of IB4-positive maxillary trigeminal ganglion neurons: Possible involvement of TREK2 activation.

IB4-positive maxillary trigeminal ganglion (TG) neurons are a subtype of afferent neurons involving nociception in orofacial regions, and excitability of these neurons is associated with orofacial nociceptive sensitivity. TREK-2 channel is a member of two-pore domain potassium (K2P) channel family mediating leak K+ currents. It has been shown previously that TREK-2 channel activity can be enhanced following GABAB receptor activation, leading to a reduction of cortical neuron excitability. In the present study, we have characterized TREK-2 channel expression on maxillary TG neurons and investigated the effect of the GABAB agonist baclofen on electrophysiological properties of small-sized maxillary TG neurons of rats. We show with immunohistochemistry that TREK-2 channels are predominantly expressed in small-sized IB4-positive maxillary TG neurons. Patch-clamp recordings on neurons in ex vivo TG preparations show that baclofen hyperpolarizes resting membrane potentials, increases outward leak currents, and decreases input resistances in IB4-positive maxillary TG neurons. Moreover, baclofen significantly reduces action potential (AP) firing in IB4-positive maxillary TG neurons. In contrast, baclofen shows no significant effect on electrophysiological properties of small-sized nociceptive-like and non-nociceptive-like maxillary trigeminal neurons that are IB4-negatve. Our results suggest that TREK-2 channel activity can be enhanced by baclofen, leading to reduced excitability of IB4-positive maxillary TG neurons. This finding provides new insights into the role of TREK-2 and GABAB receptors in controlling nociceptive sensitivity in orofacial regions, which may have therapeutic implications.