Method for treating developmental deficit induced by anesthesia

Abstract

A method for treating or reducing the severity of neurodevelopmental defects in a developing nervous system after exposure to an anesthetic by modulating or inhibiting caspase activity, for example, by administering a pan-caspase inhibitor such as pan-caspase inhibitor Q-VD-OPh to a subject having or at risk of congestive heart disease and / or having a ciliopathic disorder.

Description

[0001] METHOD FOR TREATING DEVELOPMENTAL DEFICIT INDUCED BY ANESTHESIA

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of U. S. Provisional Application No. 63 / 723,377, filed November 21, 2024 and which to the extent permitted is incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under HL139712, HL146670 and NS 127051 awarded by National Institute of Health and under W81XWH-20-1-0199 awarded by the U. S. Department of the Army. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Field of the invention. This disclosure relates to methods of treating anesthesia-associated developmental defects in pediatric or neonatal patients by administering a pan-caspase inhibitor to counter anesthetic-triggered caspase activation implicated in neurodevelopmental injury and related morphogenetic disturbances.

[0008] Description of related art. Congenital heart disease (CHD) is the most common birth defect and is associated with an increased risk for a wide range of neurodevelopmental and behavioral problems. CHD patients frequently exhibit motor skill deficits with estimates of more than half of them having moderate to severe motor development issues (1, 2). The etiology of these neurological impairments is cumulative and multifactorial, including intrinsic genetic predisposition and stressors such as neonatal cardiac surgery.

[0009] Forward recessive genetic screening performed in mouse fetuses revealed a compelling association between genes encoding proteins involved in cyclogenesis, ciliary signaling pathways and trafficking and CHD incidence (3). These findings are corroborated by clinical data, where CHD patients show a high prevalence of de novo mutations in cilia-related genes and general ciliary dysfunction (4, 5).

[0010] The primary cilium acts as a sensory organelle that regulates cellular signal transduction, thus modulating cell behavior and physiology in various tissues including brain and heart. Recent function in the developing murine heart demonstrated multiple CHD-associated heart defects, including double outlet right ventricle and atrioventricular septal defects (25). Prior work with a forebrain-specific Ift88 mutant, driven by an Emxl -dependent Cre recombinase (26), found that perinatal ethanol exposure leads to acutely elevated caspase 3 activation in pyramidal neurons of the primary motor cortex associated with long-term degenerative changes in dendritic arborization (27). Given that ethanol acts as apotent antagonist of NMDA-mediated neurotransmission(28), it is plausible that general anesthetic and sedative agents such as ketamine might interact with the loss of cilia in a comparable manner, augmenting the risk of motor impairments in patients with ciliary dysfunction such as those with CHD. To model the interaction between a sedative procedure and ciliary dysfunction we exposed the forebrain-specific Ift88 mutant mice to ketamine, followed by characterization of their motor skill learning and neuronal morphology.

[0011] Consequently, the inventors sought ways to make anesthesia safer for patients with ciliary diseases. The inventors considered that newborns could be uniquely vulnerable to anesthetic-triggered caspase activation, experience amplified neurite degeneration when cilia are impaired, and could benefit from a targeted way to interrupt the caspase cascade that underlies anesthesia-associated developmental neurotoxicity.

[0012] Based on the experimental data presented below, the inventors found that acute exposure to a single dose of ketamine during early postnatal development could lead to enhanced caspase 3 activation in layer V motor cortical pyramidal neurons of a forebrain-specific model of ciliopathy (Ift88 cKO). Neonatal ketamine exposure led to pronounced long-term fine motor skill impairments specifically in this group, which were associated with a reduction in dendritic spine density of the apical arbor of layer V motor cortical pyramidal neurons. In addition, the ketamine-treated Ift88 cKO pyramidal neurons exhibited reduced dendritic arborization complexity in adulthood. Notably, treatment with a pan-caspase inhibitor Q-VD-OPh rescued the motor learning as well as spine deficits, consistent with a key role for caspase signaling in the neuronal and behavioral alterations observed in Ift88 cKO animals. Caspase 3 activation after ketamine treatment did not lead to overt neuronal loss, arguing for a sublethal mode of augmented caspase function in this model.

[0013] BRIEF SUMMARY OF THE INVENTION

[0014] One aspect of the invention is a method for treating or reducing the severity of neurodevelopmental defects in a developing nervous system after exposure to an anesthetic by modulating or inhibiting Caspase activity, for example by administering a pan-Caspase inhibitor such as pan-caspase inhibitor Q-VD-OPh.

[0015] Another aspect is directed to increasing the safety of newborns with undeveloped nervous systems when they are subjected to anesthesia during surgery. Additionally, the methods described herein may be used to protect or treat any subject at risk of neurological damage due to negative impact of caspase activation on the brain or nervous system, for example, after prolonged surgery and anesthesia.

[0016] Another aspect of the disclosure is directed to compositions comprising pan-caspase inhibitors, carriers, and materials that increase uptake or efficacy in treating subjects who have undergone or are expected to undergo anesthesia.

[0017] Non-limiting embodiments of the disclosure include the following.

[0018] A method for preventing or treating a neurodevelopmental deficit in a subject undergoing anesthesia comprising administering at least one caspase inhibitor, wherein said method includes, but is not limited to, treatment of a child no more than 36 months of age having congenital heart disease (CHD) and / or a ciliopathic disorder, who is anesthetized with ketamine, and who is administered quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy] -methyl ketone (Q-VD-OPh) as the at least one caspase inhibitor.

[0019] The above-described method wherein the subject has CHD.

[0020] The above-described methods, wherein the subject has undergone a prolonged medical procedure under anesthesia for more than 0.25, 0.5, 1.0, 1.5, 2.0, 3.0 or 4.0 hours or any intermediate period therein.

[0021] The above-described methods, wherein the subject has a ciliopathic disorder.

[0022] The above-described methods, wherein the subject is a child no more than 6, 12, 24, or 36 months old or any intermediate age in the above range.

[0023] The above-described methods, wherein at least one caspase inhibitor is a pan-caspase inhibitor that inhibits two, three, four or more caspases.

[0024] The above-described methods, wherein the at least one caspase inhibitor inhibits caspase-3.

[0025] The above-described methods, wherein the at least one caspase inhibitor is quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy]-methyl ketone (Q-VD-OPh). The above-described methods, wherein the subject is administered ketamine during anesthesia.

[0026] The above-described methods, wherein a period of anesthesia is greater than an hour. The above-described methods, wherein the caspase inhibitor is administered intravenously, interperitoneally, or otherwise systemically.

[0027] The above-described methods, wherein the caspase inhibitor is administered intranasally, intrathecally, or otherwise directly into the brain or nervous system.

[0028] The above-described methods, wherein the anesthesia further comprises administering a muscle relaxant.

[0029] A composition comprising a caspase inhibitor and a carrier, excipient, or solvent in a form suitable for administration to a human.

[0030] The above-described composition, wherein the caspase inhibitor is present in an amount or in a concentration that inhibits caspase-3.

[0031] The above-described compositions, wherein the caspase inhibitor is present in an amount or in a concentration that inhibits multiple caspases.

[0032] The above-described compositions, wherein the caspase inhibitor comprises quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy] -methyl ketone (Q-VD-OPh) present in an amount or in a concentration that inhibits multiple caspases.

[0033] The above-described compositions, wherein the caspase inhibitor comprises is quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy] -methyl ketone (Q-VD-OPh) which is dissolved in 80-100% DMSO and / or a surfactant.

[0034] The above-described compositions that are in a form suitable for crossing the blood-brain barrier.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1A-1I. Enhanced non-apoptotic caspase 3 activation in ciliopathic motor cortex deep layer neurons following ketamine exposure.

[0036] FIG. 1A. Schematic illustrating ketamine treatment regime followed by immunohistochemical caspase signaling assessment at 16 hours post-exposure in Ift88 mutant mice. FIG. IB. Representative cleaved caspase-3 (red / inside squares and on each lateral side just below location of GC) staining pattern in a coronal section of the anterior frontal cortex of ketamine treated Ift88 cKO, with strong signal (white boxes) observed in deep layers (layer V / VI) of the primary motor cortex (PMC) among other regions.

[0037] FIG. 1C. Magnified micrographs show co-localization of cleaved caspase 3 signal with neuronal NeuN in layers V / VI of the PMC, and comparison between vehicle and ketamine treated Ift88 cHET and cKO mice.

[0038] FIG. ID. Line graph displaying mean cleaved caspase 3 (CC3) corrected total cell fluorescence (CTCF) values across 20 representative neurons / PMC region of interest (ROI) (ordered from neurons with lowest to neurons with highest mean CC3 CTCF), and their comparison between vehicle and ketamine treated Ift88 cHET and cKO mice. Note enhanced CC3 signal specifically in the ketamine treated Ift88 cKO group.

[0039] FIG. IE. Quantification of the mean CC3 CTCF across sampled deep layer neurons from groups shown in 1C-1D. (

[0040] FIG. IF. Pie charts showing proportions of neurons displaying low (0-2000), moderate (2000-6000), high (6000-10,000) and very high (10,000+) CC3 CTCF values across treatment groups.

[0041] FIG. 1G. Representative positive control (DNAse I treated) and Ift88 827 cKO (ketamine treated) TUNEL staining at 24 hours post-exposure to ketamine. Note lack of detectable TUNEL signal in ciliopathic samples.

[0042] FIG. 1H. Representative micrograph of NeuN immunoreactive neurons from adult (8-10 weeks) Ift88 cKO PMC layers V / VI.

[0043] FIG. II. Quantification of NeuN+ neurons in control and ciliopathic PMC deep layers. Note: no difference in neuronal numbers. Values in 1D-1E and I are mean ± standard error (SEM) (n=3-4 in each; 2 male + 1 female samples in Ift88 cKO (+VEH) group, 2 male + 2 female samples in other groups). *P< 0.05; ***P 833 < 0.001; ****p< 0.0001. Data in (ID) was analyzed using a 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. Data in (IE) was analyzed using a 1-way ANOVA with Tukey’s multiple comparisons test. Data in (II) was analyzed by an unpaired student’s t test. Scale bars: (IB) - 1 mm; (1C) - 50 pm; (1G) - 250 pm; (1H) - 100 pm. Abbreviations: I. P. -intraperitoneal, CC3-cleaved caspase 3, mPFC-medial prefrontal cortex, ILC-infralimbic cortex, PLC-prelimbic cortex, ACC- anterior cingulate cortex, SMC-secondary motor cortex, PMC-primary motor cortex, PSC-primary somatosensory cortex, GC-gustatory cortex, Ala-agranular insular cortex, CC-corpus callosum, OT-olfactory tubercle, aco-anterior commissure, TT -taenia tecta.

[0044] FIGS. 2A-2H. Ketamine treatment diminishes layer V neuronal arborization complexity in ciliopathic mice. (2A) Schematic showing tissue processing regime for examining layer V PMC pyramidal neuron arborization and basal spine morphometry following multiphoton imaging. (2B) Quantification of dendritic intersections, a proxy for arborization complexity, of layer V pyramidal neurons from ketamine treated controls ( Ift88 cHET) and ciliopathic ( Ift88 cKO) mice using Sholl analysis of dendrite traces obtained with Neurolucida 360. Note decreased complexity of ketamine treated Ift88 cKO neurons. (2C) Representative monochrome traces of Thyl-GFP reporter labeled layer V pyramidal neurons from control ( Ift88 cHET) and ciliopathic ( Ift88 cKO) mice (2D-2F). Quantification of basal dendritic spine density (2D), volume (2E) and contact area (2F). No differences were seen in parameters between groups. (2G) Bar graphs showing breakdown of spine class densities in the basal arbors of layer V pyramidal neurons from the ketamine treated groups. Values in (2B) and 2(D-2G) represent mean ± standard error (SEM) (n=3 in B, 2 male + 1 female samples in each group; n=3-4 for 2D-2G, 2 male + 1 female samples in Ift88 cHET (+KET) group, 2 male + 2 female samples in Ift88 cKO (+KET) group). * < 0.05. The statistical test used for (2B) was a 2- way Mixed Effects ANOVA, implemented as previously described (65). Data in (2D-2G) was analyzed by unpaired student’s t tests. Abbreviations: PMC -primary motor cortex, KET-ketamine, LP. -intraperitoneal. Comparison of neuronal soma diameter and perimeter between adult (8-10 weeks) ketamine treated Ift88 cHET and cKO mice (2H).

[0045] Figs. 3 A- 3D. Ketamine exposed Ift88 cKO mice display fine motor skill deficits.

[0046] Diagram (3 A) representing treatment regime and timeline for assessment of pellet reach fine motor skills in control ( Ifi88 cHET) and mice with forebrain-specific ciliopathy ( Ifi88 cKO). Numbers used: vehicle treated Ift88 cHET, n = 5 (3 males + 2 females); vehicle treated Ift88 cKO, n = 4 (2 males + 2 females); ketamine treated Ift88 cHET, n = 6 (3 males + 3 females); ketamine treated Ift88 cKO, n = 9 (5 males + 4 females). (B) Quantification of the fraction of successful pellet reach attempts / trial out of all reach attempts. Note significantly decreased motor learning rate in ketamine treated Ift88 cKO group. (3C) Line graph showing total number of pellet reach attempts for groups assayed in (3B). Mice from all groups did not differ significantly in pellet reach attempts. Values in (3B-3C) represent mean ± standard error (SEM). ****p < 0.0001, **P < 0.01. The statistical test used in (3B-3C) was a 2-way repeated measures ANOVA with post-hoc Tukey’s multiple comparisons test. Data in 3D was analyzed using a mixed effects model (REML) ANOVA to account for missing values due to outlier removal. Abbreviations: PRA - pellet reach assay, VEH - vehicle, KET -ketamine, PMC - primary motor cortex, CST - corticospinal tract, I. P. -intraperitoneal.

[0047] FIGS. 4A-4M. Ift88 cKO mice given ketamine have reduced baseline layer V apical spine density in the motor cortex. (4A) Diagram depicting experimental design and timeline for assessing effects of neonatal ketamine on apical dendritic spine dynamics of layer V pyramidal neurons in control Ift88 cHET) and forebrain-specific ciliopathy ( Ift88 cKO) mice containing the transgenic Thyl GFP(M) reporter allele during motor training with the accelerated rotarod. n = 3 mice per group (2 males + 1 female in both groups). (4B) Representative micrographs of layer V apical dendritic branch segments and spines tracked over the course of motor training with multiphoton imaging. White arrows show spines detected stably across several imaging sessions; red / gray arrows indicate spines lost by the next imaging session; green arrows indicate spines gained by next imaging session. (4C) Line graph showing quantification of overall dendritic spine density at baseline and following subsequent rotarod training sessions. Spine density was notably lower at baseline in ketamine treated Ift88 cKO mice compared to controls and displayed a slow increase by the final training session. (4D-4E) Bar chart comparisons of apical spine density at baseline and final training days, respectively, between experimental groups. (4F-4G) Line graphs depicting the rates of dendritic spine gain or loss, respectively, during rotarod motor training in ketamine treated control (Ift88 cHET) and forebrain-specific ciliopathy (Ift88 cKO) groups. Note continuous gain of new spines in I ft 88 cKO mice throughout training, while control spine gain plateaus following first training session. Regression trendlines are shown for each group, with P values indicating statistical significance of slope comparisons. (4H) Statistical diagram of percentages of classified dendritic spines in layer V pyramidal neurons imaged during the motor training. Spine class color mapping is shown next to the graph. (4I-4J) Bar charts displaying fraction of mushroom / mature dendritic spines at baseline and final training day, respectively. Note decrease detected in the Ift88 cKO group at the end of training. (4K-4L) Charts showing fractions of thin / immature spines detected in both groups at baseline and final training day, respectively. Note trend towards increase in thin spines on final training day in Ift88 cKO mice exposed to ketamine. (4M) Schematic summarizing effects of motor training on layer V apical spines in control and Ift88 cKO animals following perinatal ketamine administration. Values represent mean ± standard error (SEM). *P < 0.05, **P 902 < 0.01. The statistical test used in 4C was a 2-way repeated measures ANOVA. Data in 4F-4G was analyzed using regression analysis. Data in 4D-4E and 4I-4L were analyzed using unpaired student’s t tests. Abbreviations: KET - ketamine, LP.-intraperitoneal.

[0048] FIGS. 5A-5E. Perinatal treatment with pan-caspase inhibitor Q-VD-OPh suppresses neuronal caspase activation due to ketamine in Ift88 cKO mice. (5A) Schematic exhibiting ketamine and Q-VD-OPh treatment regime in perinatal Ift88 cKO animals, followed by histological assessment of caspase 3 activation in neocortical neuronal cells. (5B) Representative micrographs of primary motor cortex (PMC) layer V / VI showing magnified insets (white boxes) of cleaved caspase 3 (CC3) immunoreactivity co-localizing with neuronal NeuN in ketamine and ketamine + QVD- OPh treated Ift88 cKO mice. (5C) Quantification of the mean CC3 CTCF across sampled deep layer neurons from groups shown in 5B alongside control ketamine treated Ift88 cHET group. (5D) Line graph displaying mean cleaved caspase 3 (CC3) corrected total cell fluorescence (CTCF) values across 20 representative neurons / PMC region of interest (ROI) (ordered from neurons with lowest to neurons with highest mean CC3 CTCF), and their comparison between ketamine treated Ift88 cHET / cKO and ketamine + Q-VD-OPh treated Ift88 cKO mice. Note normalization of CC3 intensity in deep layer neurons of ketamine + Q-VD-OPh treated Ift88 cKO mice. Values show means ± standard error (SEM).

[0049]

[0050] < 0.05,****P < 0.0001. The statistical test used in 5C was a l- way ANOVA with post-hoc Tukey’s multiple comparisons test (n=3-4 each, 2 males + 2 females in cHET / cKO groups treated with ketamine and 2 males + 1 female in cKO group treated with ketamine / Q-VD-OPh). Data in 5D was analyzed using a 2-way ANOVA with post-hoc Tukey’s multiple comparisons test (n=3-4 in each). (5E) Pie charts showing proportions of neurons displaying low (0-2000), moderate (2000-6000), high (6000-10,000) and very high (10,000+) CC3 CTCF values in ketamine and ketamine + Q-VD-OPh groups (n=3-4 each). Abbreviations: CC3- cleaved caspase 3, KET-ketamine, I. P.-intraperitoneal.

[0051] FIGS. 6A-6H. Perinatal caspase signaling inhibition rescues fine motor skill and apical motor spine density due to ketamine in Ifi88 cKO mice. (6A) Schematic depicting ketamine and Q-VD-OPh treatment regime, followed by assessment of pellet reach fine motor skills or dendritic spine dynamics of layer V pyramidal neurons in ketamine and ketamine + Q-VD-OPh Ift88 cKO animals. (6B) Quantification of the fraction of successful pellet reach attempts / trial in ketamine treated groups and ketamine + Q-VD-OPh treated ciliopathic (!fi88 cKO) mice. Note significantly improved and rescued motor learning rate in ketamine + Q-VD-OPh treated Ift88 cKO group. Numbers used: ketamine treated Ift88 cHET, n = 6 (3 males + 3 females); ketamine treated Ift88 cKO, n = 9 (5 males + 4 females); ketamine + Q-VD-OPh treated Ift88 cKO, n=5 (3 males + 2 females). (6C) Representative micrographs of layer V apical dendritic branch segments and spines tracked over the course of motor training with multiphoton imaging in ketamine and ketamine + Q-VD-OPh treated Ift88 cKO groups (n = 3; 2 males + 1 female in all groups). White arrows show spines detected stably across several imaging sessions; red / gray arrows indicate spines lost by the next imaging session; green arrows (left panel) indicate spines gained by next imaging session. (6D) Line graph showing quantification of overall dendritic spine density at baseline and following subsequent rotarod training sessions. Spine density was enhanced at baseline in ketamine + Q-VD-OPh treated Ift88 cKO mice compared to group receiving only ketamine. (6E-6F) Bar chart comparisons of apical spine density at baseline and final training days, respectively, between experimental groups. The statistical tests used were 1-way ANOVA with post-hoc Tukey’s multiple comparison tests. (6G) Statistical diagram of percentages of classified dendritic spines in layer V pyramidal neurons imaged during the motor training. Spine class color mapping is shown next to the graph. (6H) Quantification of the change in thin / immature spines relative to baseline during motor learning. Note opposing trends in Ift88 cHETs and cKOs, with Q-VD-OPh treatment stabilizing thin spine turnover. (61) Line graph depicting the rates of dendritic spine gain during rotarod motor training in ketamine and ketamine + Q-VD-OPh treated ciliopathic ( Ift88 cKO) groups. Ketamine treated Ift88 cHET data is shown as a control. Note trend towards diminished gain of new spines in ketamine + Q-VD-OPh treated Ift88 cKO mice following the first training day, mimicking the control group spine gain plateau. Rates of spine loss were similar at the end of motor training in all tested groups. Values represent mean ± standard error (SEM). *P < 0.05; **P < 0.01. The statistical test used in 6B and 6D was a 2-way repeated measures ANOVA with post-hoc Tukey’s multiple comparisons test. The data in 6E-6F and 6H was analyzed using a 1-way ANOVA with post-hoc Tukey’s multiple comparisons test. Data in 61 was analyzed using regression analysis. Abbreviations: KET-ketamine, I. P.-intraperitoneal.

[0052] FIGS. 7A and 7B. Neuronal soma size does not change in ciliopathic motor cortical neurons exposed to ketamine. Comparison of neuronal soma diameter (7 A) and perimeter (7B) between adult (8-10 weeks) ketamine treated Ift88 cHET and cKO mice. Statistical testing via student’s unpaired T test. FIGS. 8A-8C. Ketamine treatment does not elicit increased microglial infiltration in the ciliopathic motor cortex. (8A) Photomicrograph showing representative immunohistochemical staining for the microglial marker Ibal at 16 hours post-ketamine treatment. (8B) Quantification of Ibal+ microglia in the deep layers of the primary motor cortex (PMS). Note no significant differences in microglial numbers due to ketamine exposure. (8C) Quantification of Ibal + microglia in PMC deep layers indicates no differences following QVD-OPh treatment. Statistical testes used were one-way ANOVA.

[0053] FIGS.9A-9H. QVD-Oph treatment effects on reaching behavior and dendritic spine class. (9A). Line graph showing total number of pellet reach attempts for groups assayed in Fig. 6B. Mice from all groups did not differ significantly in pellet reach attempts. (9B) Quantification of the fraction of pellet reach attempts resulting in a dropped pellet out of all reach attempts. The statistical test used in (A) was a 2-way repeated measures ANOVA with post-hoc Tukey's multiple comparisons test. The data in (9B) was analyzed using a mixed effects model (REML) ANOV A to account for missing values due to outlier removal. (9C) Quantification of the fraction of filopodial spines at final training day. (9D-9E) Bar charts displaying fraction of mushroom / mature dendritic spines at final training day and baseline, respectively. Note statistical trend towards a rescue effect by QVD-Oph in the Ift88 cKO group at the end of training. (9F-9G) Charts showing fractions of thin / immature spines detected in all groups at final training day and baseline, respectively. Note increase in thin spines on final training day in both Ift88 cKO groups. Statistical tests used in (C-G) were one way ANOV A. (9H) Line graph depicting the rate of dendritic spine loss during rotarod motor training in ketamine treated control ( If 88 cHET) and forebrain-specific ciliopathy ( Ift88 cKO) groups given ketamine or ketamine with QVD-Oph. Note no significant difference in the rate of spine loss during motor training as assessed by linear regression. Values in all panels represent mean± standard error (SEM).

[0054] DETAILED DESCRIPTION OF THE DISCLOSURE

[0055] The inventors sought to investigate whether anesthesia may induce developmental neurotoxicity, however, the influence of genetic predispositions associated with congenital anomalies on this toxicity remains largely unknown. Children with congenital heart disease often exhibit mutations in cilia-related genes and ciliary dysfunction, requiring sedation for their catheter or surgical interventions during the neonatal period. Here we demonstrate that briefly exposing ciliopathic neonatal mice to ketamine causes motor skill impairments, which are associated with a baseline deficit in neocortical layer V neuron apical spine density and their altered dynamics during motor learning.. These neuromorphological changes were linked to augmented non-apoptotic neuronal caspase activation. Neonatal caspase suppression rescued the spine density and motor deficits, confirming the requirement for sublethal caspase signaling in appropriate spine formation and motor learning. Our findings suggest that ciliopathy interacts with ketamine to induce motor impairments, which is reversible through caspase inhibition. Furthermore, they underscore the potential for ketamine- induced sublethal caspase responses in shaping neurodevelopmental outcomes.

[0056] Terms. The terms used herein should be given their ordinary meaning and are described in some detail below.

[0057] Ciliopathy encompasses a group of diseases characterized by abnormal formation or function of cilia which arise from mutations in genes encoding proteins essential for ciliary assembly, maintenance or function. It can produce multi-organ symptoms affecting the brain, kidneys, liver, retina, and skeletal system. Common signs or symptoms include retinal degeneration, renal dysfunctional, polydactyly, brain anomalies, skeletal abnormalities and congenital heart defects. This condition can affect motile or non-motile cilia. The methods disclosed herein may be performed on patients at risk of, or who have, a ciliopathy. Ciliopathies represent a heterogenous group of genetic disorders including Joubert syndrome, Bardet-Biedl syndrome, Meckel-Gruber syndrome and primary ciliary dyskinesia. Other recognized ciliopathies and methods for their characterization are described by, and incorporated by reference to, Focsa, I. O, et al., Clinical and genetic heterogeneity of primary ciliopathies (review), INT. J. MOL. MED.

[0058] 2021, 48(3): 176. The methods disclosed herein may be applied to prevention or treatment of developmental defects or disorders in subjects having a ciliopathy.

[0059] Diagnosis of Ciliopathy. Those skilled in the medical arts may diagnose a subject as having ciliopathy by a variety' of methods. These include genetic testing using targeted gene panels, whole-exome sequencing, or whole-genome sequencing; by detection of specific mutations using polymerase chain reaction (PCR) methods; by functional cilium phenotyping; or by deep phenotyping and bioinformatics; see the following references which are incorporated by reference and which describe these diagnostic methods: Modarage, K. et al., Molecular diagnostics of ciliopathies and insights into novel developments in diagnosing rare diseases, BR. J. BlOMED SCI.

[0060] 2022, 79, 10221. Doornbos, C. et al., Cell-based assay for ciliopathy patients to improve accurate diagnosis using ALPACA, EUR. J. HUM. GENET., 2021, 29, 1677-1689. Badano, J. L., The ciliopathies: an emerging class of human genetic disorders. ANNU REV. GENOMICS HUM GENET.

[0061] 1006, 7.'125-148; and Chen, X., et al., Phenotypic similarity for rare disease: ciliopathy diagnoses and subtyping, J. BIOMEDICAL INFORMATICS, 2019, 100. These methods may be used in conjunction with the methods disclosed herein.

[0062] Neurotoxicity more broadly refers to adverse effects on the nervous system including repair or regeneration of neurological tissue, for example, in adults or children. Such toxicity occurs in the context of anesthesia and could also occur by similar mechanisms in those who selfadminister ketamines or other anesthetic drugs. Such persons may benefit for the method disclosed herein.

[0063] Developmental neurotoxicity refers to adverse effects on a developing nervous system caused by exposure to toxic substances during prenatal and early postnatal periods and into adolescence. This term is further described by, and incorporated by reference to, COMPREHENSIVE TOXICOLOGY (Third edition), Vol. 5, 2018, 250-256.

[0064] Neonate refers to a baby within the first two weeks of birth. A newborn refers to a baby from birth to 1 to 2 months. An infant refers to a baby from 1 month to 12 months after birth. A young infant refers to a baby from 1 to 6 months of age. A toddler refers to a baby from 12 months to about three years of age. A preschooler refers to a child from 3 to 5 years of age. School age children range in age from 5 to 12 years. An adolescent or teenager ranges in age from 13-19 years of age, typically beginning with the onset of puberty. Adults range in age upward from 18-19 years. The methods disclosed herein may be performed on members of any of the above age groups.

[0065] Caspases are members of a family of cysteine proteases that specifically cleave proteins after aspartic acid residues. They play essential roles in programmed cell death (apoptosis), inflammation and various cellular regulatory processes. The Caspases described herein are human Caspases or other mammalian Caspases especially those active in humans. Caspases are produced as inactive proteins (zymogens) that become activated through dimerization and proteolytic cleavage. Caspases may exert apoptotic activity or exert non-apoptotic activities. Non-apoptotic Caspases function in relation to synaptic plasticity, specification of cell fate, cell migration, and cellular remodeling.

[0066] Apoptotic Caspases are executioners of apoptosis and help ensure the controlled and efficient elimination of cells without triggering harmful inflammation.

[0067] Some Caspases are recognized as having both apoptotic and non-apoptotic functions, such as Caspases 3, 6, 7, 8, and 9. Sublethal or localized activation of such Caspases supports development, cell migration, differentiation or tissue homeostasis while strong or sustained Caspase activation induces apoptosis.

[0068] Neonatal caspase include a family of enzymes which in a context of a fetus or neonate participate in programmed cell death or apoptosis, development and inflammation. Caspases involved in neonatal brain development include initiator Caspases such as Caspase-2, Caspase-8, Caspase-9 and Caspase- 10; effector or executioner Caspases such as Caspase-3, Caspase-6, and Caspase-7; and inflammatory Caspases including Caspase-1, Caspase 4 and 5 and Caspase-12.

[0069] Anesthesia is the medical controlled loss of sensation, awareness, and response to pain administered during surgery or other procedures that would otherwise be painful or stressful. It may be administered by mask or by an intravenous route. For some procedures it can include coadministration of a neuromuscular blocker such as atracurium or vecuronium,.

[0070] Ketamine is a cyclohexanone-derived general anesthetic with analgesic and hallucinogenic properties. It is used medically for anesthesia, pain management or to treat depression. Ketamine has two enantiomers which are S-(esketamine) and R-(arketamine). Ketamine is often a drug of choice for short-term procedures where muscle relaxation is not required. It is used as an anesthetic for minor procedures in children including treatments of cyanotic heart disease or neuromuscular disorders. Anesthesia, especially in newborns, may be required for treatment if inguinal hernia repair, tracheoesophageal fistula, congenital diaphragmatic hernia, intestinal obstruction surgery, necrotizing enterocolitis, gastroschisis and omphalocele closure, patent ductus arteriosus, congenital heart repairs, or for central line and shunt placements.. The prevention and treatment methods disclosed herein may be used in conjunction with such surgical procedures to reduce or eliminate negative effects of anesthesia. In some alternative embodiments a depolarizing e.g., succinylcholine), non-depolarizing (e.g., rocuronium, vecuronium, pancuronium,), a nondepolarizing benzylisoquinolinium, or a reversal agent such as neostigmine may be administered during anesthesia. Drugs that increase caspase activity include ketamine, stuarosporine, bortezomib, TRIAL receptor agonist, topoisomerase inhibitors (e.g., camptothecin), taxanes (e.g., paclitaxel or docetaxel), lipoic acid, carbamate and indolones, DNA damaging agents (e.g., cisplatin or etoposide), TRAIL (TNF-related apoptosis-inducing ligand), or cyclin-dependent kinase inhibitors. Exposure to such drugs may be avoided prior to anesthesia in order to further avoid neurological damage associated with activation of caspases during anesthesia.

[0071] Caspase inhibitors include inhibitors of specific caspases associated with developmental defects such as Ac-YVAD (Caspase-1), Ac-DEVD-CHO (selective for Caspase-3), Ac-IETD-CHO or Z-IETD-FMK (selective for Caspase-8), Z-LEHD FMK (selective for Caspase-9), Ac-ATS010-KE (selective for Caspase-3) and Isatin sulfonamides (selective for Caspase-3 and Caspase 7). See Dhani, S. et al., A long way to go: caspase inhibitors in clinical use, CELL DEATH Dis. 2021, 12(10):949; and Solania, A., et al., A selective and rapid cell-permeable inhibitor of human caspase-3, ACS CHEM. BIOL. 2019, 14(1 l):2463-2470, both of which are incorporated by reference.

[0072] Pan-Caspase inhibitors. Preferably, pan-Caspase inhibitors are employed in the methods disclosed herein. In some alternative embodiments, inhibitors of specific Caspases may be administered to subjects, such as those having ciliopathy and undergoing treatment with an anesthetic such as ketamine. Pan-caspase inhibitors include, but are not limited to quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), benzyloxycarbonyl-Val-Ala-Asp (Ome)-fluromethyl ketone (Z-VAD-FMK), Z-VAD(OH)-FMK, IDN-6556 or Boc-D-FMK (tert-butoxycarbonyl-aspartic acid-fluormethyl ketone, Z-D(0me)E(0me)VD-FMK, X1AP, cIAPl, and cIAP2. In some embodiments, cocktails of inhibitors that each inhibit different caspases or subsets of caspases may be formulated to provide a pan-caspase inhibitor cocktail.

[0073] Methods of prevention or treatment of developmental defects associated with anesthesia.

[0074] A pan-Caspase inhibitor, or Caspase inhibitor may be administered systemically around the time of anesthesia, for example, by intraperitoneal or intravenous administration. Advantageously such an inhibitor is selected to be capable of crossing the blood brain barrier.

[0075] Alternative modes of administration include, but are not limited to, intrathecal or intracerebroventricular delivery, intranasal administration, for example, in combination with a nanocarrier, or brain-targeted modes such as use of surfactant-coated nanoparticles, or use of liposomal or other polymeric systems. The inhibitor may be administered before, during, or immediately after anesthetic exposure to coincide with Caspase activation windows induced by exposure to an anesthetic such as ketamine. For long surgical or medical procedures such as those taking more than an hour or the inhibitor maybe repeatedly administered. It may also be administered during a post-anesthesia period. An inhibitor may be administered in a single dose or multiple doses ranging from about 0.1, 0.2, 0.4, 0.5, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9 to 10 mg / kg of body weight. Dosing may continue in a post-anesthesia period or any other period when there is continued risk of Caspase-mediated damage to the nervous system.

[0076] An inhibitor may be administered in an amount that prevents induction of apoptosis or other undesired effects by the inhibited caspase but which does not inhibit other beneficial properties of a caspase. The caspase inhibitor is administered during a therapeutically relevant period associated with, or following, anesthetic or ketamine exposure associated with caspase activation. In certain embodiments, transient administration within a post-insult interval is sufficient to attenuate or prevent caspase-mediated neurotoxicity. Complete or continuous inhibition of all caspase activity is not required; rather, inhibition to a degree that reduces injury-associated apoptotic signaling is adequate. Accordingly, a range of doses and treatment durations may be used, provided they achieve a reduction in caspase-dependent neurotoxicity during the vulnerable period following the insult. The invention therefore encompasses dosing regimens that provide partial, transient, or selective inhibition sufficient for neuroprotection.

[0077] Compositions. A pan-Caspase inhibitor, such as Q-VD-OPh, may be admixed with DMSO, ethanol and / or polyethylene glycol 400 as co-solvents prior to administration. Non-ionic surfactants, such as Tween 20 or Tween 80, may also be included. An aqueous phase such as 0.9% saline or phosphate buffered saline (PBS) may be present. Examples of suitable compositions for administration of Q-VD-OPh and other pan-Caspase or Caspase inhibitors include the following.

[0078] Co-solvent IV formulation comprising Q-VD-OPh or other inhibitor dissolved in DMSO or ethanol, diluted with PEG-400 and q.s. with sterile 0.9% saline or PBS; optionally with polysorbate 80 for dispersion of the inhibitor.

[0079] Surfactant-assisted micellar IV vehicle comprising Q-VD-OPh or other inhibitor, polysorbate 20 and / or polysorbate 80, and ethanol or propylene glycol as co-solvents, and q.s. with saline or dextrose 5% in water (D5W). See Chatzidak, M. D., et al., Advancements in Nanoemulsion-Based Drug Delivery Across Different Administration Routes, PHARMACEUTICS, 2025, 5; 17(3):337, incorporated by reference.

[0080] Liposome formulation for systemic delivery comprising hydrogenated soy phosphatidyl choline (HSPC) or DSPC and cholesterol (55:40 mol.%) optionally with DSPE-PEG2000 (5 mol.%; PEG lipid) forming unilamellar liposomes encapsulating the Q-VD-OPh in a bilayer and a buffered aqueous core. See Hu, J. et al., Pharmacokinetics and antitumor efficacy of DSPE-PEG2000 polymeric liposomes loaded with quercetin and temozolomide: Analysis of their effectiveness in enhancing the chemo sensitization of drug-resistant glioma cells, INT. J. MOL. MED. 2016, 14, 37(3): 690-702. incorporated by reference.

[0081] Ift88 cKO mice which are used in the animal models described in the Examples refer to a conditional knock out model in which the Ift88 gene which encodes intraflagellar transport protein 88 which is important for cilia formation is selectively deleted in specific tissues using Cre recombinase.

[0082] EXAMPLES

[0083] The following materials and methods were used as further described in the Examples below. Animals and treatments. Ift88 fl / fl (B6.129P2- Ift88 tmlBkyD) transgenic mice on a C57BL6 / J background were used in this study and were obtained from Jackson Laboratory (Strain #:022409; RRID: IMSR_JAX:022409). The Ift88 fl / fl line was initially crossed to Emx1-Cre (B6.129S2-Emx1tm1(cre)Krjl / Strain #:005628; RRID: IMSR_JAX:005628) to generate conditional heterozygous ( Ift88 cHET) or homozygous knockout ( Ift88 cKO) mice. The resulting animals were then crossed to the Thyl-GFP(M) line (STOCK Tg(Thyl-EGFP)MJrs / J; Strain #:007788; RRID: IMSR_JAX:007788) to generate Emxl- Cre; Ift88 fl / +; Thyl-GFP ( Ift88 cHET; Thyl-GFP) or Emxl-Cre; Ift88 fl / fl; Thyl-GFP ( Ift88 cKO; Thyl-GFP) animals for neuronal arborization and dendritic spine imaging. All animals were housed on a 12-h light / dark cycle and, unless required to be different by the experiment, provided free access to a standard rodent food pellet diet and water.

[0084] The treatment regime consisted of administering a single dose of ketamine (20 mg / kg), dissolved in 0.9% normal saline (vehicle), or vehicle intraperitoneally to postnatal day seven (P7) pups born to Emx1-Cre; Ift88 fl / +; (Thyl-GFP) x Ift88 fl / fl; (Thyl-GFP) crosses. For the caspase inhibitor experiments, a single dose of ketamine (20 mg / kg) was followed up by two doses of Q-VD-OPh (10 mg / kg) dissolved in 1% DMSO in 0.9% normal saline. The first Q-VD-OPh dose was given right after ketamine, with the second dose administered at 12-13 hours following the first.

[0085] Immunohistochemistry. To prepare histological samples for immunohistochemistry, animals at the indicated stages were intracardially perfused with ice cold phosphate buffered saline (PBS), followed by ice cold 4% paraformaldehyde (PF A) dissolved in PBS. Perfused brains were isolated and post-fixed in 4% PFA solution overnight at 4 °C. Following fixation, brains were sectioned coronally into 30 or 300 pm slices using a vibratome (Leica VT1000 S) or placed into graded sucrose solutions (15% then 30% sucrose in PBS) prior to freezing for cryosectioning into 20 pm coronal sections using a cryostat. Vibratome slices were used for free floating immunohistochemical staining for neuronal nuclear antigen (NeuN, EMD Millipore #ABN90), cleaved caspase 3 (CC3, Cell Signaling Technologies #D175), ionized calcium binding adaptor molecule 1 (Iba1, EMD Millipore MABN92) or green fluorescent protein (GFP, Abcam #ab13970). Secondary antibodies used were Alexa Fluor conjugated. Cryosections were used for slide-mounted immunohistochemistry for NeuN and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL Assay Kit, Cell Signaling Technologies #64936).

[0086] The free floating immunohistochemical procedure consisted of incubating the vibratome slices in donkey serum blocking solution (donkey serum in 0.2% Triton-X in 0.01M PBS) for 1 hour at room temperature, followed by primary antibody solutions overnight (all diluted 1:500) at 4 °C. On the following day, slices were rinsed three times for 5 minutes each with 0.01M PBS, after which they were incubated in secondary antibody solution for 2 hours at room temperature. This was followed by a final three rinses for 5 minutes each with 0.01M PBS before counterstaining with Hoechst (diluted 1:1000 in PBS, Invitrogen #33342 trihydrochloride trihydrate) and mounting in Prolong anti-fade medium (Thermofisher #P36934). Primary and secondary antibodies were diluted in donkey serum blocking solution, apart from staining involving cleaved caspase 3 antibodies, which were diluted in Immunostain Enhancer (Pierce #46644) for signal amplification.

[0087] Image analysis and quantification. Confocal images of NeuN / CC3 and Ibal staining were obtained using a Nikon A1-HD25 confocal microscope. GFP staining from Thy 1 -GFP neuronal and dendritic spine samples was imaged using a Leica SP8 confocal system. High resolution (1024 x 1024 pixels) z series scans with a 1 pm z step interval and consisting of 25-30 images were taken for NeuN / CC3 and Ibal stained slides with a 20x air objective and 3x optical zoom. Two samples of the region of interest (ROI) (one from either hemisphere) from 3 slices / animal were acquired from the primary motor cortex (PMC) deep layers (layers V-VI) for downstream analysis. The sampled coronal slices were taken from brain regions equivalent to stereotaxic coordinates +1.0mm, +1.5mm and +2mm [anteroposterior], + / -1.5mm [mediolateral] and -1.0mm [dorsoventral] relative to bregma as defined in the Allen Brain Reference Coronal Atlas for adult mice. The xy dimensions of the imaging fields of view (-300 x 300 pm) ensured that the acquired deep (layers V-VI) cortical layer ROIs were acquired in a non-overlapping manner with superficial (layers I-III) territories and were representative of PMC deep layers. The z series scans were initially flattened into single images using a maximum intensity z projection in the NIS -Elements software (Nikon). NeuN+ cells were inspected for CC3 staining, followed by marking the borders of the CC3 signal and quantification of the mean CC3 signal intensity and surface area from each NeuN+ cell. Mean background intensity was also obtained from areas surrounding the cells. To obtain corrected total cell fluorescence (CTCF) values for the CC3 signals, we used the formulas:

[0088] Integrated Density = Mean CC3 signal intensity X Mean CC3 signal area (1)

[0089] CTCF = Integrated Density - (Mean CC3 signal area X Mean background fluorescence intensity) (2)

[0090] Twenty neurons were analyzed in this manner from each ROI (for a total of -80-120 neurons / animal), prior to averaging for comparisons between groups.

[0091] For GFP stained slides, coronal slices (300 pm) from two independent section levels anterior to the bregma were imaged and individual neurons were used for morphological reconstruction and analysis with Neurolucida 360 software. High resolution (1024 x 1024 pixels) z series scans were obtained using either a 0.5 pm or 1 pm step interval for basal dendritic spines and neuronal arbors respectively. Confocal z stacks consisted of 40-60 or 100- 150 images for spine and neuronal arbor imaging respectively. To maximize the number of basal spines and dendrites captured for morphometric analyses, tile scans were performed with 4 x 4, or 5 x 5 z tiles stitched together using 10% overlap with the Leica LAX software. Dendritic spines were imaged using an oil immersion lOOx objective and a 2.5x optical zoom, while neuronal arbors were captured using a 20x air objective and 2.5x optical zoom. Tile z scans were imported into Neurolucida 360 (MBF) for three-dimensional neuronal arbor and dendritic spine tracing, and spine classification using default parameters. Once completed, individual traces were analyzed using Neurolucida Explorer (MBF) to obtain measures of branching complexity, soma volume, spine density, spine volume and spine class frequency.

[0092] Cranial window surgeries. Following treatment at P7, transgenic mice ( Ift88 cHET; Thyl-GFP and Ift88 cKO; Thyl-GFP) underwent chronic cranial window implantation surgeries at 2-3 months of age, as previously described(62), with slight modifications. Briefly, animals were anesthetized using a cocktail of ketamine / xylazine (100 mg / kg ketamine, 16 mg / kg xylazine, administered intraperitoneally) and placed on a body warmer pad (Kent Scientific #RT-0520) kept at 37 °C. The toe pinch reflex and breathing pattern were used to monitor the depth of anesthesia. Carprofen (5 mg / kg) and dexamethasone (2.5 mg / kg) were administered subcutaneously to minimize inflammation and brain edema. The scalp was shaved using a trimmer and eye ointment applied to prevent corneal drying. The animal was mounted on the stereotaxic instrument (Kopf Model #902) and 1% lidocaine was applied subcutaneously under the scalp. A single vertical incision was made over the skull, and the skin and connective tissue were retracted to expose the frontal skull bone. A 3- 3.5 nun diameter circular craniotomy was performed over the motor cortex (craniotomy center at ~1 mm anterior and 1 mm lateral to bregma), using a micro drill and regularly cooling the drill site with ice cold 0.9% saline solution.

[0093] Absorbable hemostatic gelatin sponges (Fisher Scientific #NC0654350) dipped in ice cold 0.9% saline were used to stop and clear up any minor bleeding during the procedure. Following careful removal of the skull bone flap, a custom circular coverslip assembly (5 mm outer and 3 mm inner diameter) was used to replace the bone with a glass “window” as previously described(62). The glass window was glued to the surrounding bone using an instant adhesive (Loctite Super Glue liquid). A custom-built metal headpiece (Xometry) was then glued to the surrounding bone carefully to avoid contacting the glass window. Dental cement (Lang #1334CLR) was applied to the edges of the craniotomy and exposed areas surrounding the headpiece. Following this, the surgical animals were kept on the warming pad until they regained mobility.

[0094] Multiphoton spine imaging. Surgical mice were allowed at least one week to recover from the procedure before imaging. To train animals on a motor task, we employed the accelerated rotarod. The mice were initially introduced to the rotarod apparatus by placing them on a fixed speed (5 revolutions / min) regime for 15 minutes on the first day of testing. The mice were then trained on an accelerated rotarod for the next four days (5-40 revolutions / min over 5 minutes), for three trials / day, with trials separated by 5 minutes. All training trials were video recorded using a Basler GigE camera mounted on a tripod and Noldus Etho Vision XT software. Trials were performed at the same time of the day to limit behavioral variations due to circadian rhythm fluctuations. Video recordings were scored by 584 a blinded investigator. The rotarod acclimation and training sessions (apart from first training 585 session) were followed up (-2 hours) by daily imaging and tracking of individual apical dendritic 586 spines in neocortical layer I, using a multiphoton microscope (Evident FVMPE-RS). Briefly, the mouse was anesthetized using a cocktail of ketamine / xylazine (2 / 3 of surgical dose) and placed on a warming pad kept at 37 °C. Head fixation was achieved by bolting the metal headpiece to custom- built holders printed three-dimensionally in ABS polymer (Xometry). Imaging was performed using a 25x water immersion multiphoton objective (Evident XLPLN25XWMP2) and a 3x or 10x optical zoom. To capture GFP fluorescence a Tksapphire Mai Tai femtosecond laser (Spectra Physics) was tuned to 950 nm for excitation, and the emitted light was collected with a high sensitivity GaAsP detector. A z series scan of a larger field of view was initially obtained at 3x optical zoom for an overview of the area of interest and storing of coordinates for repetitive imaging.

[0095] Higher resolution (800 x 800 pixels) z scans of groups of dendritic spines were acquired at 10x optical zoom using a 0.5 pm z step interval and 50-100 images for each region of interest (ROI). Five ROIs were acquired and tracked for each animal in each imaging session (for a total of four imaging sessions / animal). Image acquisition was performed using the Fluoview software (Evident).

[0096] The resultant z series images were analyzed using Fiji / ImageJ to obtain apical spine density, spine class frequency and to track spine turnover (total of -550 spines) during motor training. Spine classification was performed using spine diameter, length and length / diameter ratio parameters as described previously(42).

[0097] Pellet reach assays. To assess fine motor skill acquisition, we employed the pellet reach assay in Emxl-Cre; Ift88fl / + ( Ift88 cHET) and Emxl-Cre; Ift88 fl / fl ( Ift88 cKO) mice. Mice are capable of rapidly learning the task, involving forelimb extension to reach a food pellet, pronated grasping of the pellet and finally forelimb retraction until the pellet is consumed. Deficits in successful performance of the task have been associated with impairments of the motor cortex due to stroke (63), or experimental manipulation of motor cortical circuit activity through chemo- and optogenetic means (64). To motivate pellet reaching behavior, mice were initially fasted for 5 days by restricting food to elicit a 10-15% body weight loss, which was maintained during training trials. Following the fasting period individual mice were placed in a Plexiglass training chamber with a single vertical slit located on the front wall. Individual food pellets were placed in front of the slit opening at a distance which requires the animals to use their forelimb to reach the pellet. A successful reaching attempt was termed successful if the mouse could reach, grasp the pellet, and consume it. Missed or dropped pellets were labelled as unsuccessful reaching attempts. Individual mice were assayed for five days, with each daily trial lasting for 20 minutes or until the animal successfully reached 20 pellets, whichever came first. All training sessions were performed at the same time of the day to limit behavioral variations due to circadian rhythm fluctuations and were video recorded using a Basler GigE camera mounted on a tripod and Noldus EthoVision XT software. Trial recordings were scored by a blinded investigator.

[0098] Statistical analysis. All statistical analyses were performed in GraphPad Prism 8. All data are represented as mean ± standard error (SEM) from at least three biological replicates for each experiment. Cleaved caspase 3 intensity and spine density comparisons between three or more groups were analyzed by one- way ANOVA. Two factor data such as Sholl analyses were analyzed with two-way ANOVA followed by post-hoc Tukey’s or Sidak’s tests. Comparisons between two independent groups were made using unpaired t-tests or non-parametric Kruskal- Wallis tests. Spine dynamics and motor learning performance data were analyzed using repeated measures two-way ANOVA tests followed by post-hoc Tukey’s multiple comparisons tests. Differences between groups were judged to be statistically significant when p < 0.05. Asterisks denote statistical significance p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

[0099] EXAMPLE 1. Caspase 3 activation in layer V ciliopathic motor neurons exposed to ketamine 110 does not cause apoptosis.

[0100] The inventors initially set out to assess the potential for interaction between ketamine exposure and neuronal ciliary deficits during the early murine postnatal window. To determine the effect of neonatal ketamine on caspase activation in neurons of the ciliopathic neocortex, a single dose of ketamine (20 mg / kg) or vehicle was administered intraperitoneally to P7 pups (equivalent to human neonate) born to crosses between Emxl-Cre; Ift88 fl / + and Ift88 / / / / mice (Fig. 1 A). This ketamine dose was chosen since prior work demonstrated it to be the minimal subanesthetic dose necessary for eliciting a subtle yet significant elevation in cleaved caspase 3 (CC3) immunoreactivity in the caudate putamen brain area, and deemed to be equivalent to a sedating / subanesthetic ketamine dose for an infant human(2 ). In this way we endeavored to evaluate whether ciliopathy augments ketamine-induced CC3 activation. CC3 signal intensity' was assessed in neocortical neurons at 16 hours following treatment, using signal co-localization between NeuN and CC3.

[0101] The inventors observed strongly elevated neuronal CC3 immunoreactivity in the ketamine-treated Ift88 cKO group, forming a specific pattern across the perinatal neocortex (Fig. 1B-1C). This result corroborated previous findings using ethanol-exposed Ift88 mutant animals(27), which showed increased caspase 3 activation in neurons of the primary motor cortex (PMC), specifically in ciliopathic Ift88 cKO neurons exposed to ethanol. Caspase 3 activation was enhanced in sparse populations of neurons in the medial prefrontal (mPFC) and primary somatosensory cortex (PSC), while strikingly elevated in deeper layers of the PMC and across the gustatory cortex (GC) (Fig. IB). The pattern of elevated caspase activation in the ketamine treated ciliopathic group ( Ift88 cKO + KET) was not uniform among deep layer neurons with neighboring cells displaying contrasting levels of signal (Fig. 1C). This heterogeneity in caspase activation is intriguing as it suggests variable sensitivity to ketamine among neurons occupying the same cortical layers and / or functional specialization, regardless of their ciliary integrity.

[0102] Quantification of mean CC3 corrected total cell fluorescence (CTCF) revealed a highly significant increase in caspase 3 activation among ciliopathic ( Ift88 cKO) ketamine-exposed deep layer neurons (Fig. ID). Interestingly, while a higher CC3 signal was present in Ift88 cKO neurons across a range of CC3 intensities, the neurons with higher CC3 CTCF values showed a highly statistically significant CC3 signal enhancement compared to neurons in other groups. This difference was also reflected in the mean CC3 CTCF value comparisons (Fig. IE) as well as the proportions of low, moderate, or high CC3 intensity neurons present in layer V / VI primary motor cortex (Fig. IF). Vehicle treated Ift88 cHET and cKO mice did not show differences in CC3 intensity suggesting that the loss of primary cilia alone does not influence signaling through caspase 3 in perinatal neocortical neurons. On the other hand, ketamine treatment did not result in CC3 elevation in Ift88 cHET neurons, indicating that a single dose of ketamine does not have an observable impact on caspase signaling in neurons expressing functional primary cilia.

[0103] Caspase 3 activation has been classically associated with the initiation of apoptosis and cell death, which culminates with the process of DNA fragmentation. To assess DNA fragmentation in ketamine treated Ift88 cKO neurons we performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 24 hours following ketamine administration. The inventors chose this time window based on prior research in rat models of focal cerebral ischemia, which showed substantial DNA fragmentation in the cortex and striatum at 24 hours post-reperfusion injury(jO). Ciliopathic ketamine-exposed neocortical neurons did not display detectable TUNEL immunoreactivity, in stark contrast to DNAse I-treated positive control tissue (Fig. 1G). To follow up and confirm this finding, we examined neuronal numbers in PMC deep layers of adult (8-10 weeks) Ift88 cHET and cKO ketamine treated animals (Fig. 1H). Quantification of NeuN immunoreactive cells in the deep layers of the PMC did not show a statistically significant difference in neuronal number between control and ciliopathic brains (Fig. II). A lack of neuronal loss in the ciliopathic PMC suggests that the augmented neuronal caspase signaling, observed shortly after ketamine exposure, does not lead to apoptosis and long-term cell loss. This finding agrees with a multitude of studies demonstrating non-apoptotic, sub-lethal roles for caspase signaling in both physiological and pathological contexts (12).

[0104] These results point to a potential causative correlation between loss of primary cilia and acute ketamine exposure in driving enhanced caspase 3 activation in neocortical deep layer motor neurons, whereas neither factor is sufficient on its own for the observed changes.

[0105] EXAMPLE 2. Ciliopathic layer V motor neurons exposed to ketamine have reduced arbor 166 complexity.

[0106] Cleaved caspase 3 is known to destabilize microtubule networks in neurons, leading to reduced arborization and enhanced dendritic pruning (31, 32). To investigate whether the augmented caspase signaling observed following ketamine exposure influences dendritic arborization in our experimental groups, we crossed Thyl-GFP reporter mice with the Emxl-Cre; Ift88 flox lines, followed by ketamine treatment at P7. Brain tissue was collected and histologically processed from animals that had undergone rotarod motor training and multiphoton imaging (Fig.

[0107] 2A). Arborization complexity analysis using the Sholl method revealed a statistically significant reduction in branching complexity in ketamine exposed Ift88 cKO pyramidal neurons (Fig. 2B-2C). Analyses of neuronal soma size did not show clear differences by genotype (Fig. 2H), suggesting that only the ciliopathic dendritic compartment was affected by the ketamine treatment (FIGS. 7A and 7B). These data argue that caspase 3 activation reduces the branching complexity of ciliopathic layer V pyramidal neurons (Fig. 2C), signifying structural neuronal changes rather than apoptotic cell loss.

[0108] Caspase signaling within neurons is often accompanied by neuroinflammatory changes in the local microenvironment. One of the principal drivers of neuroinflammation are microglial cells (33), which infiltrate the injury site / s for the phagocytosis of dying cells and cellular debris and neuroprotection. Microglia have also been demonstrated to engage in presynaptic trogocytosis, a process of partial engulfment of intracellular material through cell-to-cell contacts 34). Despite a lack of conclusive evidence for a microglial role in alterations to postsynaptic spine elements, these previous findings argue that microglia do remodel synaptic contacts in the postnatal brain. Thus, we examined microglial numbers in the deep layers of the PMC corresponding to the areas 188 where CC3+ neurons were detected, using Ibal immunoreactivity in the ketamine treated Ift88 cHET and cKO groups (FIG. 8A). We did not detect statistically significant differences in the total numbers of microglia between groups, suggesting that neonatal ketamine treatment does not elicit increased microglial infiltration in the ciliopathic group (FIG. 8B).

[0109] Caspase signaling can elicit changes in neuronal dendritic spines alongside alterations to the arborization pattern. To establish whether perinatal ketamine affects the basal dendritic compartment, we performed spine tracing, morphometry and classification on basal dendritic branches imaged from the same samples obtained for dendritic arborization studies (Fig. 2A). We did not detect differences in mean basal spine density' (Fig. 2D), spine volume (Fig. 2E) or spine contact area (Fig. 2F) between control and ciliopathic ketamine-treated groups. In addition, basal spine classification and comparison did not show significant changes in the density of individual immature and mature spine classes (Fig. 2G), although there was a trend towards an increase in stubby spine density in the Ift88 cKO group.

[0110] In summary, our morphometric analyses of layer V pyramidal neurons revealed a reduction in arborization complexity of ciliopathic neurons following ketamine exposure, without observable differences in neuronal cell numbers or basal spine density and morphology. This finding suggests that ketamine-induced caspase signaling results in long-term remodeling of basal dendrites of maturing layer V ciliopathic pyramidal neurons.

[0111] EXAMPLE 3. Ift88 cKO mice exposed to ketamine display fine motor deficits. Extensive non-apoptotic caspase 3 activation was observed in cilia-deficient motor cortical neurons and was associated with morphological changes. To investigate the functional consequences of the observed increase in neuronal caspase signaling after ketamine treatment in Ift88 cKO animals, we assessed fine motor skill acquisition in our experimental cohorts. Fine motor skill development is known to be deficient in individuals with CHDs after neonatal cardiac surgery(35, 36). Forelimb extension and food pellet reaching is a robust and well-validated fine motor skill assay in rodents (37). Ift88 cHET and cKO mice treated with either vehicle or ketamine at P7 were left to mature prior to fasting at 8-10 weeks of age to ensure adequate food motivation (Fig. 3A).

[0112] In correlation with the immunohistochemical data on caspase activation (Fig. 1), we observed a strongly reduced success rate at the pellet reach task solely in the ketamine-exposed Ift88 cKO group (Fig. 3B). On the other hand, ketamine treated Ift88 cHET animals and the vehicle groups exhibited no change in motor skill acquisition. This discrepancy in motor performance of the ketamine h eated Ift88 cKO mice could not be explained by lack of motivation in the ciliopathic animals, given that their mean total reaching attempts were significantly higher compared to cHET experimental groups at later stages (Fig. 3C). These data pointed to the possibility that elevated cleaved caspase 3 might be responsible for structural changes in deep layer neurons that lead to impairments in fine motor skill learning.

[0113] EXAMPLE 4. Ift88 cKO mice given ketamine have reduced apical spine density’ in layer V 227 motor pyramidal neurons.

[0114] Pyramidal neuron dendritic spine turnover has been closely associated with the process of memory consolidation, behavioral flexibility, and learning such as motor skill learning (38, 39). Indeed, spine content and plasticity in neocortical motor pyramidal neurons are directly correlated with rodent performance on the accelerated rotarod task, which is impaired in contexts of reduced spine plasticity' such as during ageing (40, 41). Given that caspase 3 activity' is known to influence the pruning of dendritic branches and spines in a non-apoptotic manner(37), we investigated spine density and dynamics of mature layer V motor pyramidal neurons in ketamine treated cohorts. To assess possible dendritic spine changes after ketamine-induced non-apoptotic caspase activation and the effect on motor skill learning, we performed chronic cranial window surgeries on ketamine treated control ( Ift88 cHET; Thyl-GFP(M)) and ciliopathic ( Ift88 cKO; Thyl-GFP(M)) mice, followed by multiphoton imaging and tracking of dendritic spines on the accelerated rotarod (Fig.

[0115] 4A). The Thyl-GFP(M) transgenic reporter allows for sparse, yet bright labeling of individual dendritic segments and spines in apical arbors of neocortical layer V pyramidal neurons (Fig. 4B).

[0116] Individual dendritic segments located in neocortical layer I were tracked during training, and their spine content and morphology were examined. Overall dendritic spine density was strongly reduced in the Ift88 cKO group at baseline conditions, prior to the onset of accelerated rotarod training, and it displayed a slow increase up until the final training day (Fig. 4C-E). Control spine density remained at a higher steady level through most training days, displaying a subtle decline by the end of the experiment. This finding is consistent with the interpretation that a single dose of neonatal ketamine treatment reduces apical spine density in ciliopathic pyramidal neurons and leads to a dampened motor learning rate during the initial skill acquisition phase.

[0117] The inventors next examined the dynamics of individual spines between imaging sessions, by quantifying the proportion of gained or lost spines relative to their baseline numbers (Fig. 4F-4G).

[0118] The control group dendrites experienced a -23% gain in spines between baseline and first trial imaging sessions, which plateaued and declined by the final training day. In contrast, the ciliopathic dendrites matched the initial rate of spine gain yet continuously gained new spines until the end of motor training, displaying a mean ~51 % increase in gained spines compared to baseline levels (Fig. 4F). To examine the relationship between spine gain and time more closely we performed linear regression analysis on data obtained between trial days 1 and 3. The rate of spine gain displayed by Ift88 cKO animals during this time window was significantly higher compared to controls, indicating enhanced spine addition in ciliopathic mice (Fig. 4F). In contrast, the rate of spine loss did not differ between the two conditions (Fig. 4G) indicating an overall net spine gain in ketamine-exposed ciliopathic layer V neurons during training. These results point to continuous addition of new spines during motor learning in the ciliopathic group leading up to the final rotarod training day, whereas control dendrites exhibit a more balanced spine turnover profile, favoring spine loss in the later training phases.

[0119] To understand whether motor training had differential impacts on different dendritic spine classes, individual spines were classified using morphological parameters, as described previously(42). Overall, we observed all major spine classes in both experimental groups across all imaging sessions, including mushroom, filopodial, thin and stubby spines (Fig. 4H). Branched spines were extremely rare, with only a single observation. Ciliopathic apical dendrites displayed a trend towards an inverted frequency profile of mushroom and thin spines compared to controls, with a tendency towards decrease or increase respectively, by the end of motor training. Mushroom spines, which represent mature postsynaptic sites forming stable synapses, did not differ between groups in their proportions at baseline (Fig. 41). However, by the final training day their proportion decreased by -40% in ciliopathic mice (Fig. 4J). Thin spines, which are transient and associated with learning, exhibited greater variability within groups, and while baseline differences are unclear (Fig. 4K), their mean proportion displayed an increase (-82%) in ciliopathic animals in the final imaging sessions (Fig. 4L). These findings indicate that while motor training impacts spine turnover in both groups, ciliopathic mice exhibit a spine profile characteristic of continuous learning which is protracted compared to controls, which acquire a more mature spine profile and diminish their spine plasticity by the end of training.

[0120] In summary, our spine dynamics profiling experiments show evidence of decreased baseline apical spine density in ciliopathic layer V pyramidal neurons exposed to ketamine (Fig.

[0121] 4M). Consistent with a delay in fine motor skill learning (Fig. 3), ciliopathic neurons displayed protracted spine plasticity during motor training, with a delay in acquisition of mature postsynaptic sites by the final training session.

[0122] EXAMPLE 5. Pharmacological inhibition of caspase signaling silences caspase activation in 286 ciliopathic mice due to ketamine.

[0123] To confirm the involvement of caspases in ketamine-induced protracted spine plasticity and motor learning impairments in ciliopathic mice, the inventors utilized quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy] -methyl ketone (Q-VD-OPh), a non-competitive inhibitor of caspases 1, 3, 8, and 9, which can cross the blood-brain barrier and displays no overt toxicity in vivo(43). To confirm effective inhibition of caspase 3 activation in our model, we administered two doses of Q-VD-OPh (10 mg / kg each, intraperitoneally) with the first dose given immediately following ketamine treatment, and the second dose 12 hours after the first (Fig. 5 A). As in the initial experiments (Fig. 1), CC3 immunoreactivity was assessed in conjunction with NeuN in the deep layers of the PMC. Treatment with Q-VD-OPh completely suppressed neuronal CC3, bringing signal to below control ( Ift88 cHET+ ketamine) levels ( -70% reduction) (Fig. 5B-C). This reduction was reflected across different signal intensities with particularly lower CTCF values among more strongly immunoreactive neurons (Fig. 5D-E). While Q-VD-OPh treatment strongly reduced caspase 3 activation due to ketamine in the Ift88 cKO motor cortex, it resulted in a more subtle reduction trend (-16.5%) in Ibal+ microglial cells (Supp. Figure S3C), which was not statistically significant. These findings indicate that a short treatment regime with a pan-caspase inhibitor, administered promptly after ketamine exposure, is sufficient to rescue neuronal caspase 3 activation in the deep layers of the ciliopathic PMC.

[0124] EXAMPLE 6. Caspase suppression rescues fine motor deficits and apical spine density in 305 Ift88 cKO motor cortex.

[0125] To investigate the consequences of dampened caspase 3 activation on fine motor skills and dendritic spines of motor layer V pyramidal neuron apical dendrites, we employed the same experimental paradigm used during neonatal ketamine exposure (Fig. 6A). One set of cohorts was fasted for pellet reach fine motor skill acquisition, while the other underwent chronic cranial window implantation over the motor cortex, followed by accelerated rotarod training and concurrent spine multiphoton imaging (Fig. 6A).

[0126] Brief Q-VD-OPh treatment given following ketamine-induced acute caspase activation resulted in the complete rescue of fine motor skill acquisition on the pellet reach task, represented by the enhanced success rate of pellet acquisition in Ift88 cKO mice that matched controls (ketamine-treated Ift88 cHETs) (Fig. 6B). This finding strongly argues that the initial upregulation in neuronal caspase 3 activation following ketamine exposure is critical for the fine motor skill deficit seen in ciliopathic Ift88 cKO animals. 318

[0127] Given that the observed fine motor skill acquisition delay in ketamine treated Ift88 cKOs 319 was associated with altered apical spine density and turnover in layer V pyramidal neurons of the 320 PMC, we assessed Q-VD-OPh treated spines during motor learning (Fig. 6C). Quantification of 321 overall spine density revealed a complete rescue in Q-VD-OPh treated mice at baseline, and a return to control levels (Fig. 6D-6E). After the final training day, spine density did not differ significantly between the experimental groups (Fig. 6F). This result indicates that suppressing caspase 3 activation following neonatal ketamine sedation is sufficient to restore apical spine density to control levels, and that exacerbated caspase signaling mediates apical spine pruning with resultant density deficits that persist into adulthood. Spine classification at baseline and during motor learning trials revealed a tendency towards reduction in filopodial spines in Q-VD-OPh treated mice across all imaging sessions compared to ketamine exposed Ift88 cKOs (Fig.

[0128] 6G and Fig. 9C). This reduction resulted in overall levels of filopodial spines that mimicked controls (ketamine treated Ift88 cHETs). Mushroom and thin spine fractions also exhibited a propensity towards a return to control levels at final trial day and baseline respectively (Figs. 9D-9G. Thin immature spines exhibited an increase in proportion at the final trial day relative to baseline in ciliopathic mice, which was significantly different to the relative decrease seen in controls (Fig. 6H). Q-VD-OPh normalized this elevation in thin spines, although this was not statistically significant. However, the thin spine fraction in Q-VD-OPh treated animals remained at higher overall levels than controls in the final imaging session (Fig. 9F), arguing that this component of the spine phenotype is more dependent on the ciliopathy than ketamine- induced caspase 3 activation.

[0129] To understand whether Q-VD-OPh treatment alters apical spine turnover during motor learning, we quantified the fraction of gained and lost spines as before. Q-VD-OPh restored the spine gain profile in Ift88 cKO animals to that seen in controls, likely due to the normalization of baseline spine density (Fig. 61). Linear regression analysis found no statistically significant difference in the rate of spine loss between all experimental groups (Fig. 9G). Overall, our findings indicate that caspase-3 activation, due to acute neonatal ketamine exposure in ciliopathic animals, is a key driver of reduced apical spine density and altered spine turnover in layer V pyramidal neurons of the adult PMC. Brief pharmacological inhibition of caspase signaling following ketamine administration is capable of completely restoring apical spine density and fine motor skill acquisition to control levels, while stabilizing spine turnover rates during motor learning.

[0130] The work described by the Examples above, demonstrates for the first time that neonatal exposure to a single dose of ketamine is capable of inducing caspase 3 with sub-lethal yet detrimental consequences on neuronal structure and spine plasticity in ciliopathic pyramidal neurons, leading to motor learning impairments. While a complete loss of primary cilia is not frequently encountered in patient populations, apart from rare genetic conditions such as primary ciliary dyskinesia (44), genetic variants with damaging effects on cilia integrity and function are frequent in patients with CHD (3, 45, 46). Anesthesia and sedation are currently critical components of care in infants with CHD for various surgical and / or interventional procedures (47, 48). Given the increased frequency of motor skill impairments in individuals with CHD and the high prevalence of mutations associated with ciliary dysfunction, our findings suggest potentially harmful interactions between anesthetic exposures and genomic variants associated with primary cilia deficits in the CHD population. In addition, our results imply a capacity for acute ketamine sedation to induce caspase-dependent, non-apoptotic neuronal structural changes long-term, which to our knowledge has been underappreciated in the field. Notably, we demonstrate that brief treatment with the pan-caspase inhibitor Q-VD-OPh, known to possess low in vivo toxicity, rescues the delay in motor learning and deficits in neuronal structure and spine plasticity, revealing brief inhibition of caspases as a potential therapeutic target for children with CHD who require multiple sedation procedures during the early postnatal period.

[0131] Prior work by Ishii et al. ( 7) demonstrated that perinatal ethanol exposure causes caspase-induced degenerative changes to neuronal arbors in ciliopathic Ift88 cKO mice, which occur due to downregulation of the Akt pathway. Pharmacological activation of insulin-like growth factor 1 (IGF1)-Akt signaling was shown to restore dendritic arborization in the ciliopathic model. Interestingly, caspase 3 activation was present after ethanol treatment in neurons where ciliary loss was induced from P5, suggesting the adverse interaction occurs primarily within the perinatal window, rather than from earlier embryonic loss of cilia such as occurs with the Emxl-Cre driver. Activation of the IGF1 receptor was localized at the base of the primary cilium, near the ciliary transition zone, a diffusion barrier controlling the trafficking of ciliary proteins, which is particularly enriched for damaging de novo variants in cases of CHD (4, 49). Our study extends these results to ketamine, another NMDA receptor antagonist, with likely shared mechanistic features with ethanol exposure in a ciliopathic context.

[0132] Complementary to the ethanol findings, we detected a specific subpopulation of layer V neurons susceptible to ketamine-induced caspase 3 activation, pointing to possible differences in susceptibility to environmental agents among neighboring cells. Neuronal degeneration and death are often accompanied by neuroinflammatory infiltration by astrocytic and microglial cells(50); however, we did not detect robust changes in microglia following ketamine administration in ciliopathic mice. One possible explanation is that the sampled time point (16 hours post- treatment) is too early to detect the full extent of microglial expansion. Alternatively, the level of neuronal caspase 3 activation in our model does not reach a critical threshold which would trigger robust neuroinflammatory changes in the PMC. Indeed, the observed augmentation of caspase signaling might be elevated enough to trigger cytoskeletal fragmentation and pinning of dendrites and spines, yet not so high to cause neuronal death. Whether inhibitor of apoptosis (IAP) proteins specifically limit the progression to neuronal apoptosis in the ciliopathic context is unknown and would benefit from future study. Our motor skill behavioral analysis detected robust impairments of fine motor skill learning in the ciliopathic animal group following a single dose of ketamine. Since we did not observe evidence of reduced motivation in ketamine treated Ift88 cKOs, it is plausible that either components of motor initiation or integration are impaired in the ciliopathic mice. Curiously, visuomotor integration is a particularly vulnerable behavioral domain in patients with CHD (51, 52), which could be at risk due to adverse interactions between anesthetics / sedatives and susceptible genetic backgrounds. Concurrently with the fine motor skill impairments, ciliopathic mice treated with ketamine showed a striking reduction in apical spine density and protracted spine turnover profiles, structural correlates of reduced synaptic plasticity and delayed learning. Interestingly, the spine density deficit was confined to the apical rather than basal compartment, 404 although this might be explained by the fact that we assessed basal dendritic spines at the end of motor training, rather than throughout learning. Apical spine class assessments showed a tendency towards fewer mature spines and a greater fraction of immature spines by the end of motor training in ciliopathic mice after ketamine, indicating delayed plasticity and consequently motor learning. Repeated exposures to ketamine between the second and third postnatal week have previously been shown to dampen spine plasticity in adulthood (53). Given the observed caspasedependent 410 spine density impairment at the onset of motor training, the likely explanation for the protracted spine turnover profile is the propensity for adding new immature spines coupled with a paucity' of mature, motor memory-consolidating spines. Whether the spine density defect occurred primarily due to initial caspase-dependent pruning(54) following ketamine or was also shaped secondarily by altered spinogenesis and spine dynamics prior to training(55) is not clear and would be an interesting avenue to investigate further.

[0133] In addition to the spine deficits, we found evidence of a loss of dendritic complexity in ciliopathic pyramidal neurons after a single ketamine exposure, which coupled with the spine impairment points to substantial caspase-driven morphological changes resulting due to an interaction between ciliary dysfunction and ketamine exposure. Prior studies examining the antidepressant effects of ketamine in wild-type rats showed that acute ketamine treatment increased short-term prefrontal cortical neuron dendritic arborization and spine density, likely via enhanced mammalian target of rapamycin (mTOR) signaling (56, 57). These discrepancies could reflect differing time windows of ketamine exposure, as well as the fact that our study investigated the interaction with a loss of primary cilia. In contrast with previous work which focused more on the classical caspase-driven apoptosis pathway, our study suggests non-apoptotic, long term cytomorphological changes could be a cause for concern in patient populations receiving anesthesia that are genetically predisposed to ciliary impairments or carry IGF1-Akt pathway polymorphisms (58), such as in CHD(5, 59, 60).

[0134] Given that caspases are ubiquitous housekeeping enzymes during development, long-term caspase inhibition potentially has off-target side effects. Thus, we evaluated a potent pan-caspase inhibitor, Q-VD-OPh, during a limited period that specifically targets caspase activation during / immediately after neonatal ketamine administration. Our results indicate that a brief exposure to a pan-caspase inhibitor is sufficient to correct the motor behavior impairments and spine deficits induced by ketamine. Q-VD-OPh use has not been associated with toxicity in vivo and has shown efficacy in limiting caspase activation in models of stroke, spinal cord injury and neurodegenerative disorders(73). In our model the inhibitor rescued both the fine motor skill learning rate and the baseline apical spine density, pointing to caspase-dependent pruning as the likely cause of the behavioral impairment. Caspase inhibition also restored the fraction of mature motor spines, possibly aiding the process of earlier memory consolidation compared to ciliopathic mice exposed to neonatal anesthesia. Given the safety profile of Q-VD-OPh and its efficacy in inhibiting both initiator and effector caspases with brief treatment, it shows promise for limiting caspase-dependent neuronal structural impairments due to agents such as sedatives and general anesthetics.

[0135] While this work identifies a clear interaction between ketamine and loss of primary cilia in the neonatal neocortex, there are several limitations. Firstly, complete ablation of primary cilia in a systemic manner is incompatible with life, and severe ciliopathies such as Meckel-Gruber syndrome lead to perinatal lethality (67). The ciliopathic mouse model allows for targeted loss of primary cilia in excitatory neurons of the neocortex from late mid-gestation, providing the means to evaluate cell-specific sensitivity to the interaction between ketamine and ciliary loss. Partial ciliary loss, primary cilia shortening and / or aberrant function are more frequently encountered among patients, and future modelling efforts taking this into account would help resolve whether this detrimental interaction occurs in more common clinical scenarios. Secondly, given that ketamine signals through many of the same pathways as most inhalable anesthetics, determining whether similar interactions occur between agents such as sevoflurane and primary cilia impairments would be highly pertinent. In addition, neonatal anesthesia for major surgery such as neonatal CHD repair typically involves a mix of compounds including benzodiazepines, propofol, sevoflurane combined with fentanyl and dexmedetomidine, which in combination might result in caspase- induced apoptosis and cell death, rather than sublethal structural remodeling as we have observed. Evaluating the interaction between these compounds and damaging ciliary variants would be feasible the mouse and would yield information on compound-specific effects on neuronal morphology and motor function. The ketamine dose, while relatively low, points to heightened sensitivity of ciliopathic animals to ketamine-induced sedation, which would likely be exacerbated by higher, continuously administered doses encountered clinically. Thirdly, exploring the mechanistic basis of the sublethal caspase effects on neuronal spines, and how this signaling cascade is triggered by ketamine exposure is necessary in future studies to understand how caspase inhibition might mediate its neuroprotective effects on neuronal structure and function.

[0136] To conclude, the work disclosed herein demonstrates that even a single exposure to ketamine sedation can induce detrimental, caspase-driven morphological changes to motor pyramidal neurons as well as motor learning deficits in ciliopathic subjects. The caspase-mediated effects are non-apoptotic and reversible through early and brief caspase inhibition.

[0137] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0138] As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0139] It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0140] As used herein, the word "include," and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms "can" and "may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features.

[0141] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items and may be abbreviated as “ / .” As used herein in the description, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “substantially,” “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and / or position to indicate that the value and / or position described is within a reasonable expected range of values and / or positions. For example, a numeric value may have a value that is + / - 0.1% of the stated value (or range of values), + / - 1% of the stated value (or range of values), + / - 2% of the stated value (or range of values), + / -5% of the stated value (or range of values), + / - 10% of the stated value (or range of values), + / -15% of the stated value (or range of values), + / - 20% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[0142] Disclosure of values and ranges of values for specific parameters (such as temperatures, molecular weights or weight percentages) are not exclusive of other values and ranges of values useful herein. It is envisioned that two or more specific exemplified values for a given parameter may define endpoints for a range of values that may be claimed for the parameter. For example, if Parameter X is exemplified herein to have value A and also exemplified to have value Z, it is envisioned that parameter X may have a range of values from about A to about Z. Similarly, it is envisioned that disclosure of two or more ranges of values for a parameter (whether such ranges are nested, overlapping or distinct) subsume all possible combination of ranges for the value that might be claimed using endpoints of the disclosed ranges. For example, if parameter X is exemplified herein to have values in the range of 1-10 it also describes subranges for Parameter X including 1-9, 1-8, 1-7, 2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 as mere examples. A range encompasses its endpoints as well as values inside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2, 3, 4, <5 and 5.

[0143] The terms “we” and “our” refer to the inventors.

[0144] As used herein, the words "preferred" and "preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

[0145] Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.

[0146] The description and specific examples, while indicating embodiments of the technology, are intended for purposes of illustration only and are not intended to limit the scope of the technology. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations of the stated features. Specific examples are provided for illustrative purposes of how to make and use the compositions and methods of this technology and, unless explicitly stated otherwise, are not intended to be a representation that given embodiments of this technology have, or have not, been made or tested.

[0147] To the extent any material is stated to be “incorporated by reference,” such incorporation is intended to be effective only as permitted under the applicable law and rules of the relevant Office.

[0148] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, especially referenced is disclosure appearing in the same sentence, paragraph, page or section of the specification in which the incorporation by reference appears.

[0149] The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the technology disclosed herein. Any discussion of the content of references cited is intended merely to provide a general summary of assertions made by the authors of the references, and does not constitute an admission as to the accuracy of the content of such references.

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Claims

CLAIMS1. A method for preventing or treating a neurodevelopmental deficit in a subject undergoing anesthesia comprising administering at least one caspase inhibitor,wherein said method includes, but is not limited to, treatment of a child no more than 36 months of age having congenital heart disease (CHD) and / or a ciliopathic disorder, who is anesthetized with ketamine, and who is administered quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy] -methyl ketone (Q-VD-OPh) as the at least one caspase inhibitor.

2. The method of claim 1 wherein the subject has CHD.

3. The method of claim 1, wherein the subject has undergone a prolonged medical procedure under anesthesia for more than an hour.

4. The method of claim 1, wherein the subject has a ciliopathic disorder.

5. The method of claim 1, wherein the subject is a child no more than 36 months old.

6. The method of claim 1, wherein the subject is a child no more than 24 months old.

7. The method of claim 1, wherein at least one caspase inhibitor is a pan-caspase inhibitor.

8. The method of claim 1, wherein the at least one caspase inhibitor inhibits caspase-3.

9. The method of claim 1, wherein the at least one caspase inhibitor is quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy]-methyl ketone (Q-VD-OPh).

10. The method of claim 1, wherein the subject is administered ketamine to induce or maintain anesthesia.

11. The method of claim 1, wherein a period of anesthesia is greater than an hour.

12. The method of claim 1, wherein the caspase inhibitor is administered intravenously, intraperitoneally, or otherwise systemically.

13. The method of claim 1, wherein the caspase inhibitor is administered intranasally, intrathecally, or otherwise directly into a brain or nervous system.

14. The method of claim 1, wherein the anesthesia further comprises administering a muscle relaxant.

15. A composition comprising a caspase inhibitor and a carrier or excipient in a form suitable for administration to a human.

16. The composition of claim 15, wherein the caspase inhibitor is present in an amount or in a concentration that inhibits caspase-3.

17. The composition of claim 15, wherein the caspase inhibitor is present in an amount or in a concentration that inhibits multiple caspases.

18. The composition of claim 15, wherein the caspase inhibitor comprises quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy] -methyl ketone (Q-VD-OPh) present in an amount or in a concentration that inhibits multiple caspases.

19. The composition of claim 15, wherein the caspase inhibitor comprises quinolyl-valyl-O-methylaspartyl-[-2,6- difluorophenoxy] -methyl ketone (Q-VD-OPh) which is dissolved in 80-100% DMSO and / or a surfactant.

20. The composition of claim 15 in a form suitable for crossing a blood-brain barrier.

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