Enhancer AAVs for targeting spinal motor neurons and descending motor pathways in rodents and macaque.

Experimental access to cell types within the mammalian spinal cord is severely limited by the availability of genetic tools. To enable access to lower motor neurons (LMNs) and LMN subtypes, which function to integrate information from the brain and control movement through direct innervation of effector muscles, we generated single cell multiome datasets from mouse and macaque spinal cords and discovered putative enhancers for each neuronal population. We cloned these enhancers into adeno-associated viral vectors (AAVs) driving a reporter fluorophore and functionally screened them in mouse. The most promising candidate enhancers were then extensively characterized using imaging and molecular techniques and further tested in rat and macaque to show conservation of LMN labeling. Additionally, we combined enhancer elements into a single vector to achieve simultaneous labeling of upper motor neurons (UMNs) and LMNs. This unprecedented LMN toolkit will enable future investigations of cell type function across species and potential therapeutic interventions for human neurodegenerative diseases.

Striosomes Target Nigral Dopamine-Containing Neurons via Direct-D1 and Indirect-D2 Pathways Paralleling Classic Direct-Indirect Basal Ganglia Systems.

Balanced activity of canonical direct D1 and indirect D2 basal ganglia pathways is considered a core requirement for normal movement, and their imbalance is an etiologic factor in movement and neuropsychiatric disorders. We present evidence for a conceptually equivalent pair of direct-D1 and indirect-D2 pathways that arise from striatal projection neurons (SPNs) of the striosome compartment rather than from SPNs of the matrix, as do the canonical pathways. These S-D1 and S-D2 striosomal pathways target substantia nigra dopamine-containing neurons instead of basal ganglia motor output nuclei. They modulate movement oppositely to the modulation by the canonical pathways: S-D1 is inhibitory and S-D2 is excitatory. The S-D1 and S-D2 circuits likely influence motivation for learning and action, complementing and reorienting canonical pathway modulation. A major conceptual reformulation of the classic direct-indirect pathway model of basal ganglia function is needed, as well as reconsideration of the effects of D2-targeting therapeutic drugs.

Buccal Fat Advancement-Transposition Flap for Reconstruction of Midface Volume Defects.

Background: The buccal fat pad (BFP) has previously been utilized for repair of various defects of the head and neck. Objectives: We explore the utility of a pedicled buccal fat advancement-transposition (BFAT) flap in various forms of midface reconstruction through a variety of surgical approaches and characterize its volume and axial reach in human anatomic specimens. Methods: Ten adult full-head human anatomic specimens were dissected, and a single surgical case demonstrating the use of a BFAT flap is described. Results: Nasolabial, subciliary, and deep plane facelift incisions all provided access to the BFP for use as a BFAT flap. The mean volume of mobilizable fat contained within a BFAT flap accessible through external incision was 7.1 cm3. Once fully mobilized, the externalized BFAT flap had a mean axial reach of 6.9 cm without tension. We also present a case illustrating the successful use of a BFAT flap for volumization of a large midface defect secondary to Mohs micrographic surgical resection of a cutaneous malignancy. Discussion: The BFAT flap, which exhibited substantial volume and reach in this study, can be harvested through multiple dissection windows or pre-existing defects and be used to reconstruct a variety of midface defects.

Morpho-electric diversity of human hippocampal CA1 pyramidal neurons.

Hippocampal pyramidal neuron activity underlies episodic memory and spatial navigation. Although extensively studied in rodents, extremely little is known about human hippocampal pyramidal neurons, even though the human hippocampus underwent strong evolutionary reorganization and shows lower theta rhythm frequencies. To test whether biophysical properties of human Cornu Amonis subfield 1 (CA1) pyramidal neurons can explain observed rhythms, we map the morpho-electric properties of individual CA1 pyramidal neurons in human, non-pathological hippocampal slices from neurosurgery. Human CA1 pyramidal neurons have much larger dendritic trees than mouse CA1 pyramidal neurons, have a large number of oblique dendrites, and resonate at 2.9 Hz, optimally tuned to human theta frequencies. Morphological and biophysical properties suggest cellular diversity along a multidimensional gradient rather than discrete clustering. Across the population, dendritic architecture and a large number of oblique dendrites consistently boost memory capacity in human CA1 pyramidal neurons by an order of magnitude compared to mouse CA1 pyramidal neurons.

Sinonasal Symptom Correlation With the Postoperative Polyp Scale (POPS).

BACKGROUND: Commonly used endoscopic nasal polyp grading scales have been shown to correlate poorly with symptom scores and quality of life metrics. The recently described Postoperative Polyp Scale (POPS) is a grading system that more accurately characterizes polyp recurrence in postoperative sinus cavities by describing incremental recurrence in relation to the surgically opened sinus cavities. OBJECTIVE: The objective of this study was to determine if the POPS correlated with sinonasal symptoms. METHODS: CRSwNP patients were prospectively administered SNOT-22 questionnaires and graded according to the POPS starting at their 1-month postoperative appointments. Total POPS scores (sum of each side) and Max POPS score (larger value of left and right) were correlated with SNOT-22 total scores and subdomains using Kendall correlation testing. RESULTS: A total of 127 patients were enrolled in the study. Both Total POPS or Max POPS were significantly correlated to the SNOT-22 total score (P < .001, P < .001), Rhinologic (P < .001, P < .001), Extra-Nasal Rhinologic (P < .001, P < .001), Ear/Facial (P < .001, P < .001), and Psychologic (P = .028, P = .017) subdomains. Kendall's tau indicated strong correlation (≥0.3) with Rhinologic subdomain, moderate correlation (.21-.29) with Extra-Nasal Rhinologic and Ear/Facial subdomains, and weak correlation (.1-.19) with Psychologic subdomain. CONCLUSION: Previous endoscopic nasal polyp grading scales poorly correlate with symptoms and patient reported outcome measures. The new POPS moderately correlates with the total SNOT-22 score and strongly correlates with the Rhinologic subdomain, indicating that it may have good potential as a tool to evaluate postoperative CRSwNP patients.

Development of the 12-Item Facial Complaints Evaluation Scale (FaCES-12).

OBJECTIVES: Chronic rhinosinusitis and related rhinologic disorders are common in routine otolaryngologic practice. Common presenting symptoms include nasal obstruction, facial pain, facial pressure, headache, and a subjective feeling of the face feeling "swollen," a perceptual distortion. No validated scale exists to assess facial pain in addition to perceptual distortion or headache. The objective was to develop a novel scale for assessment of facial symptoms experienced by patients presenting for rhinologic evaluation. METHODS: This was a prospective validation cross-sectional study. A patient questionnaire, the 12-item Facial Complaints Evaluation Scale (FaCES-12), was created to evaluate facial symptoms based on clinical experience and the literature, including severity and timing of facial pain, facial pressure, facial perceptual swelling, and headache. Each item was assessed utilizing an 11-point Likert scale ranging from 0 to 10 in severity. Data was collected prospectively from 210 patients in 1 private and 2 academic otolaryngologic practices from August to December 2019 along with the PROMIS Pain Intensity Scale 3a and 22-Item Sino-nasal Outcome Test. Construct validity was determined using Pearson correlation and exploratory factor analysis. Internal consistency and test-retest reliability were assessed by calculating Cronbach's alpha and assessing test-retest scores. RESULTS: A new 12-item scale named FaCES-12 was developed. FaCES-12 demonstrated high reliability with a Cronbach's alpha of .94 and high test-retest reliability (r = .90). The scale revealed very strong correlation with the PROMIS Pain Intensity Scale 3a (r = .81) and moderate correlation with the Sino-nasal Outcome Test (r = .48). Exploratory factor analysis demonstrated the scale contained interrelated variables that measured unique components of facial sensations. CONCLUSION: The FaCES-12 is a valid and reliable instrument for use in the evaluation of facial symptoms. Further research into the application of this scale is warranted.

Preseptal transconjunctival approach in orbital floor fracture repairs to prevent postoperative lower eyelid malposition.

PURPOSE: Lower eyelid malposition can be a complication following orbital floor fracture surgeries. We present our incidence of lower eyelid malposition from a large case series of orbital floor fracture repairs using the 'swinging eyelid' approach and 'hang back' technique. METHODS: A retrospective review of all orbital fracture surgeries at our institution from November 2011 to March 2021 was performed. Primary outcomes included the incidence of lower eyelid malposition by category, the average time to presentation after primary surgery, and reoperation rates among cases with lower eyelid complications. RESULTS: A total of 438 cases that involved repair of the fractured orbital floor were identified. Six patients (1.37 %) developed lower eyelid malposition following primary orbital floor repair. Two patients (0.46 %) developed reverse ptosis of the lower eyelid. Two patients (0.46 %) returned with lower lid cicatricial ectropion. One patient (0.23 %) had postoperative lower eyelid retraction. One patient (0.23 %) had postoperative lower eyelid cicatricial entropion. No cases of lower lid flattening, lower eyelid fat flattening, or eyelid notch was noted. All patients with lower eyelid malposition underwent additional surgeries except one patient with reverse ptosis (83.3 %). The average time to the presentation of postoperative complications from the surgery date was 292.8 days (range = 49 days to 3.5 years). CONCLUSION: Lower eyelid malposition after orbital floor repair is a known complication that can be decreased by employing the 'swinging eyelid' with a preseptal approach and closure by the 'hang back' technique.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Microscale visualization of cellular features in adult macaque visual cortex.

Expansion microscopy and light sheet imaging enable fine-scale resolution of intracellular features that comprise neural circuits. Most current techniques visualize sparsely distributed features across whole brains or densely distributed features within individual brain regions. Here, we visualize dense distributions of immunolabeled proteins across early visual cortical areas in adult macaque monkeys. This process may be combined with multiphoton or magnetic resonance imaging to produce multimodal atlases in large, gyrencephalic brains.

Expansion-assisted selective plane illumination microscopy for nanoscale imaging of centimeter-scale tissues.

Recent advances in tissue processing, labeling, and fluorescence microscopy are providing unprecedented views of the structure of cells and tissues at sub-diffraction resolutions and near single molecule sensitivity, driving discoveries in diverse fields of biology, including neuroscience. Biological tissue is organized over scales of nanometers to centimeters. Harnessing molecular imaging across three-dimensional samples on this scale requires new types of microscopes with larger fields of view and working distance, as well as higher imaging throughput. We present a new expansion-assisted selective plane illumination microscope (ExA-SPIM) with diffraction-limited and aberration-free performance over a large field of view (85 mm 2 ) and working distance (35 mm). Combined with new tissue clearing and expansion methods, the microscope allows nanoscale imaging of centimeter-scale samples, including entire mouse brains, with diffraction-limited resolutions and high contrast without sectioning. We illustrate ExA-SPIM by reconstructing individual neurons across the mouse brain, imaging cortico-spinal neurons in the macaque motor cortex, and tracing axons in human white matter.

Frontal Sinus Anatomy Variations in Race and Sex Using the International Frontal Sinus Anatomy Classification.

Objective: To identify frontal sinus anatomical variations grouped by patient sex, race, and presence of chronic rhinosinusitis with frontal sinus involvement (CRFS) using the International Frontal Sinus Anatomy Classification (IFAC) system. Methods: A retrospective review from 2015 to 2020 was performed of consecutive adult patients with computed tomography sinus imaging. Prevalence of frontal sinus cells using the IFAC system was recorded. Comparisons were made between patient race, sex, and CRFS groups. Results: A total of 184 patients (368 sides) were included, 90 (48.9%) of which had CRFS. The racial distribution was 50 white (27.2%), 50 black (27.2%), 45 Hispanic/Latino (24.5%), and 39 Asian (21.2%) patients. The supra agger cell was most prevalent in the white population (P = 0.009), and supraorbital ethmoid cells were more prevalent in the Asian population (P = 0.017). Patients with frontal sinus disease were more likely to have a supraorbital ethmoid cell (P = 0.024). Overall, CRFS was more prevalent in the Asian population (P = 0.013). Conclusion: Significant differences in frontal sinus anatomy and disease exist between patient race and sex. Supraorbital ethmoid cells are associated with the development of CRFS. These patterns in frontal sinus anatomy should be noted prior to frontal sinus surgery to improve surgical awareness and outcomes.

Adeno-associated viral vectors for functional intravenous gene transfer throughout the non-human primate brain.

Crossing the blood-brain barrier in primates is a major obstacle for gene delivery to the brain. Adeno-associated viruses (AAVs) promise robust, non-invasive gene delivery from the bloodstream to the brain. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates. Here we report on AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques, which has improved delivery efficiency in the brains of multiple non-human primate species: marmoset, rhesus macaque and green monkey. CAP-Mac is neuron biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques and is vasculature biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver functional GCaMP for ex vivo calcium imaging across multiple brain areas, or a cocktail of fluorescent reporters for Brainbow-like labelling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. As such, CAP-Mac is shown to have potential for non-invasive systemic gene transfer in the brains of non-human primates.

Genes associated with cognitive ability and HAR show overlapping expression patterns in human cortical neuron types.

GWAS have identified numerous genes associated with human cognition but their cell type expression profiles in the human brain are unknown. These genes overlap with human accelerated regions (HARs) implicated in human brain evolution and might act on the same biological processes. Here, we investigated whether these gene sets are expressed in adult human cortical neurons, and how their expression relates to neuronal function and structure. We find that these gene sets are preferentially expressed in L3 pyramidal neurons in middle temporal gyrus (MTG). Furthermore, neurons with higher expression had larger total dendritic length (TDL) and faster action potential (AP) kinetics, properties previously linked to intelligence. We identify a subset of genes associated with TDL or AP kinetics with predominantly synaptic functions and high abundance of HARs.

Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates.

Delivering genes to and across the brain vasculature efficiently and specifically across species remains a critical challenge for addressing neurological diseases. We have evolved adeno-associated virus (AAV9) capsids into vectors that transduce brain endothelial cells specifically and efficiently following systemic administration in wild-type mice with diverse genetic backgrounds, and in rats. These AAVs also exhibit superior transduction of the CNS across non-human primates (marmosets and rhesus macaques), and in ex vivo human brain slices, although the endothelial tropism is not conserved across species. The capsid modifications translate from AAV9 to other serotypes such as AAV1 and AAV-DJ, enabling serotype switching for sequential AAV administration in mice. We demonstrate that the endothelial-specific mouse capsids can be used to genetically engineer the blood-brain barrier by transforming the mouse brain vasculature into a functional biofactory. We apply this approach to Hevin knockout mice, where AAV-X1-mediated ectopic expression of the synaptogenic protein Sparcl1/Hevin in brain endothelial cells rescued synaptic deficits.

Target cell-specific synaptic dynamics of excitatory to inhibitory neuron connections in supragranular layers of human neocortex.

Rodent studies have demonstrated that synaptic dynamics from excitatory to inhibitory neuron types are often dependent on the target cell type. However, these target cell-specific properties have not been well investigated in human cortex, where there are major technical challenges in reliably obtaining healthy tissue, conducting multiple patch-clamp recordings on inhibitory cell types, and identifying those cell types. Here, we take advantage of newly developed methods for human neurosurgical tissue analysis with multiple patch-clamp recordings, post-hoc fluorescent in situ hybridization (FISH), machine learning-based cell type classification and prospective GABAergic AAV-based labeling to investigate synaptic properties between pyramidal neurons and PVALB- vs. SST-positive interneurons. We find that there are robust molecular differences in synapse-associated genes between these neuron types, and that individual presynaptic pyramidal neurons evoke postsynaptic responses with heterogeneous synaptic dynamics in different postsynaptic cell types. Using molecular identification with FISH and classifiers based on transcriptomically identified PVALB neurons analyzed by Patch-seq, we find that PVALB neurons typically show depressing synaptic characteristics, whereas other interneuron types including SST-positive neurons show facilitating characteristics. Together, these data support the existence of target cell-specific synaptic properties in human cortex that are similar to rodent, thereby indicating evolutionary conservation of local circuit connectivity motifs from excitatory to inhibitory neurons and their synaptic dynamics.

Ostial Patency Measurements After Endoscopic Sphenoidotomies and Frontal Sinusotomies.

BACKGROUND: Sphenoid and frontal sinuses have narrow ostia and are prone to stenosis. However, their relative rates of patency are not well established, and descriptive rates of sphenoid stenosis have never been reported. The objective is to measure the patency of the sphenoid and frontal sinus ostia postoperatively. METHODS: A prospective multi-institutional cohort study was performed. Ostial patency was measured at surgery and 3 and 6 months postoperatively. Pertinent clinical history such as the presence of nasal polyps and prior history of ESS as well as the use of steroid eluting stents were recorded. Overall stenosis rates were calculated for both the sphenoid and frontal sinuses, and Wilcoxon-Signed Rank Test was used to compare intraoperative and postoperative ostial areas. Factorial Analysis of Variance (ANOVA) was performed to determine effects of 5 clinical factors. RESULTS: Fifty patients were included. The mean sphenoid sinus ostial area decreased 42.2% in size from baseline to 3 months postoperatively (T0 55.2 ± 28.7 mm vs T3 m 31.8 ± 25.5 mm, P < .001). The mean frontal sinus ostial area decreased 39.8% in size from baseline to 3 months postoperatively (T0 33.7 ± 17.2 mm vs T3 m 19.9 ± 15.1 mm, P < .001). Neither the sphenoid nor the frontal sinus ostial patency demonstrated statistically significant change from 3 to 6 months postoperatively. CONCLUSION: Both sphenoid and frontal sinus ostia routinely narrow postoperatively, predominately from baseline to 3 months. These findings can serve as a reference for both clinical outcomes and future studies of these surgeries.

Comparative single cell epigenomic analysis of gene regulatory programs in the rodent and primate neocortex.

Sequence divergence of cis- regulatory elements drives species-specific traits, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains to be elucidated. We investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset, and mouse with single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome, and chromosomal conformation profiles from a total of over 180,000 cells. For each modality, we determined species-specific, divergent, and conserved gene expression and epigenetic features at multiple levels. We find that cell type-specific gene expression evolves more rapidly than broadly expressed genes and that epigenetic status at distal candidate cis -regulatory elements (cCREs) evolves faster than promoters. Strikingly, transposable elements (TEs) contribute to nearly 80% of the human-specific cCREs in cortical cells. Through machine learning, we develop sequence-based predictors of cCREs in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Lastly, we show that epigenetic conservation combined with sequence similarity helps uncover functional cis -regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Postoperative Polyp Scale (POPS): Development of a New Sinonasal Polyp Grading Scale.

OBJECTIVE: Commonly used endoscopic grading scales, such as the nasal polyp scale, inadequately describe the degree of polyposis found postoperatively in the paranasal sinus cavities. The purpose of this study was to create a novel grading system that more accurately characterizes polyp recurrence in postoperative sinus cavities, the Postoperative Polyp Scale (POPS). METHODS: A modified Delphi method was utilized to establish the POPS using consensus opinion among 13 general otolaryngologists, rhinologists, and allergists. Postoperative endoscopy videos from 50 patients with chronic rhinosinusitis with nasal polyps were reviewed by 7 fellowship-trained rhinologists and scored according to the POPS. Videos were rated again 1 month later by the same reviewers, and scores were assessed for test-retest and inter-rater reliability. RESULTS: Overall inter-rater reliability for the first and second reviews of the 52 videos was Kf = 0.49 (95% CI 0.42-0.57) and Kf = 0.50 (95% CI 0.42-0.57) for the POPS. Intra-rater reliability showed near-perfect test-retest reliability for the POPS with Kf = 0.80 (95% CI 0.76-0.84). CONCLUSION: The POPS is an easy-to-use, reliable, and novel objective endoscopic grading scale that more accurately describes polyp recurrence in the postoperative state which will be useful in the future for measuring the efficacy of various medical and surgical interventions. LEVEL OF EVIDENCE: 5 Laryngoscope, 133:2885-2890, 2023.

Intravenous functional gene transfer throughout the brain of non-human primates using AAV.

Adeno-associated viruses (AAVs) promise robust gene delivery to the brain through non-invasive, intravenous delivery. However, unlike in rodents, few neurotropic AAVs efficiently cross the blood-brain barrier in non-human primates (NHPs). Here we describe AAV.CAP-Mac, an engineered variant identified by screening in adult marmosets and newborn macaques with improved efficiency in the brain of multiple NHP species: marmoset, rhesus macaque, and green monkey. CAP-Mac is neuron-biased in infant Old World primates, exhibits broad tropism in adult rhesus macaques, and is vasculature-biased in adult marmosets. We demonstrate applications of a single, intravenous dose of CAP-Mac to deliver (1) functional GCaMP for ex vivo calcium imaging across multiple brain areas, and (2) a cocktail of fluorescent reporters for Brainbow-like labeling throughout the macaque brain, circumventing the need for germline manipulations in Old World primates. Given its capabilities for systemic gene transfer in NHPs, CAP-Mac promises to help unlock non-invasive access to the brain.