Ischemic diseases, the leading cause of disability and death, are caused by the restriction or blockage of blood flow in specific tissues, including ischemic cardiac, ischemic cerebrovascular and ischemic peripheral vascular diseases. The regeneration of functional vasculature network in ischemic tissues is essential for treatment of ischemic diseases. Direct delivery of pro-angiogenesis factors, such as VEGF, has demonstrated the effectiveness in ischemic disease therapy but suffering from several obstacles, such as low delivery efficacy in disease sites and uncontrolled modulation. In this review, we summarize the molecular mechanisms of inducing vascular regeneration, providing the guidance for designing the desired nanomedicines. We also introduce the delivery of various nanomedicines to ischemic tissues by passive or active targeting manner. To achieve the efficient delivery of nanomedicines in various ischemic diseases, we highlight targeted delivery of nanomedicines and controllable modulation of disease microenvironment using nanomedicines. Blood vessels mediating the balance of nutrients, oxygen and waste products, are one of the most important organ systems throughout the body in vivoVarious strategies have been developed to induce new vessel formation. Delivery of cytokines, such as VEGF, has been considered as an efficient strategy to induce angiogenesis Myocardial infarction (MI) is mainly caused by the stenosis and occlusion of the coronary artery, which leads to inadequate blood supply to the cardiac tissues and subsequently myocardial hypoxia. It was estimated that coronary atherosclerosis caused ~12 million deaths annually due to either ST-segment elevation MI  or non-ST-segment elevation MI  ex vivo and then in dogs in 1988 The most common used myocardial ischemia model in laboratory is the permanent ligation of the left anterior descending artery (LAD) Ischemic stroke is caused by transient or permanent occlusion of a cerebral blood vessel eventually leading to brain infarction in vivo experiment model in stroke research is the intraluminal suture middle cerebral artery occlusion (MCAO) model, which does not need craniectomy Obviously, ischemic stroke is highly heterogeneous and complicated, which leads to experimental models of stroke can only represent specific aspects of this complex disease Hindlimb ischemia is commonly caused by narrowing or blockage of arteries supplying blood to the lower limbs The most frequently used hindlimb ischemia model is performed in murine, wherein surgical ligation of the femoral artery at a specific site triggers arteriogenesis in femoral collaterals proximally and angiogenesis in distal ischemic muscle Figure 1): i) thrombus formation phase. Under some physiological and pathological factors, the vessels are occluded as a result of the thrombus formation, which in turn results in the deficient supply of the oxygen and nutrients; ii) cell damage phase (day 0 to 2). After 0-2 days, the accompanying of ischemic events such as hypoxia, hypoperfusion, metabolic dysregulation induce cell apoptosis and necrosis; iii) inflammatory phase (day 0 to 7). The inflammatory features appear. Following the secretion of cytokines, chemokines and growth factors from damaged and inflammatory cells, the neutrophil, monocyte, and lymphocyte home and migrate into the necrotic tissues for the removal of dead cells. In addition, the inflammatory events induce metalloproteinases (MMPs) expression for the degradation of extracellular matrix (ECM) Once occurrence of vascular occlusion, the surrounding tissues undergo a continuum of molecular, cellular, and extracellular responses. In general, the responses can be divided into four phases , Ang-2, PDGF, and stem cell factor (SCF) Ischemia-induced angiogenesis is a physiological response to tissue hypoxia. At Oet al. showed that adeno-associated virus (AAV) encoded HIF-1α obviously induced capillary sprouting by injection into skeletal muscle, whereas AAV-VEGF only induced endothelial proliferation without proper vessel formation et al. also found that despite marked induction of hypervascularity, transgenic mice overexpressing HIF-1α did not induce edema, inflammation, or vascular leakage, which commonly occurred in VEGF-overexpressed transgenic mice et al. also displayed that, in addition to superior angiogenic effect, HIF-1α activation can avoid common problems relevant with overexpression of individual angiogenic growth factors, such as formation of unstable/leaky vasculature.The advantages of mastering hypoxia for angiogenesis have been demonstrated by accumulated evidences. Pajusola e.g., VEGF) that directly stimulate vessel formation or maturation is no doubt major strategies for ischemic therapy. Although the approach has showed the positive outcomes in various studies, its disadvantages are also apparent Throughout the previous reports, delivery of isolated angiogenic growth factors  controllable directionality of angiogenesis by localized delivery of angiogenic signaling at a target site . Whereas nanoparticles enable fine-tuned release of growth factors over time, this strategy is largely dependent on particle properties, which is not adequately suitable for the complex microenvironment in sequential process of ischemic disease. Instead, ischemic microenvironment-responsive drug delivery may represent a more advanced way to achieve personalized therapy.Moreover, it is critical to spatio-temporally guide an angiogenic response, i.e. control when and where it is induced, for how long and in what direction. Nanoparticle mediated drug delivery provides: i) a long-lasting drug release through the long-term retention time of nanoparticles; ii) a controlled release manner to avoid unwanted side effects, such as ectopic angiogenesis, vascular leakage, tumor formation, Table  nanomedicines that rationally designed for targeting of ischemic tissues by passive and active manner can alleviate the ischemic cascade events, such as oxidative stress, inflammatory, cell death and tissue repair. Typically, the high permeability of blood vessels in ischemic tissue allows the accumulation of nanomedicines to release the payloads, thereby promote vascular regeneration and alleviate inflammatory responses in various ischemic diseases . For the dissociation of the cargo from the nanomedicines, we herein do not summarize owing their extensively discussion in many reviews.A functional vascular system is essential for the proper maintenance of tissues function by delivering oxygen and nutrients, removing waste, and trafficking the transport of immune cells. The interruption of adequate blood causes rapid damage in the ischemic tissue, leading to cell death unless blood supply recovers quickly. Therefore, enhanced neovascularization is essential to establish an equilibrium between energy supply and demand in the ischemic tissues to prevent cell death and tissue dysfunction. Additionally, injured cells in the ischemic tissue can release intracellular chemokines or cytokines which initiate the process of inflammatory response. It is worth noting that the ischemic cascade events occur in a certain sequence and influence one another. According to the reports presented in When ischemic diseases occur, ischemia leads to vascular dysfunction, one manifestation of which is increased vascular permeability. In recent years, nanoparticles have been used to improve drug delivery to ischemic tissues because of their unique physicochemical properties and their controllable particle size. After intravenous injection, nanoparticles accumulate in damaged tissues through EPR effect. The longer the nanoparticles circulate in the blood, the greater the probability that nanoparticles reach the ischemic area and cross the leaky vessels. Thus, passive diffusion is the main approach for nanoparticles to accumulate in the ischemic lesions. In an ischemic hindlimb model, animal imaging showed that the accumulation of graphene oxide-iron oxide nanoparticles in ischemic tissues reached saturation at day 3 and decreased gradually et al. designed liposomes by co-assembling indocyanine green for photoacoustic imaging and CREKA  peptide for fibrin targeting, which exhibited great capacity to accumulate in the cardiac infarct region 2S nanodots was prepared, which showed high affinity for injured endothelial cells overexpressing AT1R While passive targeting relies on the leakiness of blood vessels, active targeting can be achieved by conjugating ligands on the surface of nanoparticles, enabling site-specific interactions and improving its retention in the target tissues rather than other organs. Of note, the engineered nanomedicines for active targeting cannot avoid passive targeting owing to the enhanced vascular permeability in ischemic tissues. In most ischemic tissue, the disruption of tissue physiology creates an abnormal local environment which can be harnessed for drug delivery. NPs can be modified to respond to specific tissue microenvironment such as low pH and hypoxia et al. reported that neutrophil membrane coated magnetic nanoprobes, composed of PLGA nanoparticles loaded with superparamagnetic iron oxide nanoparticles, exhibited selective targeting to activated endothelial cells in a rodent model of transient middle cerebral artery occlusion et al. fabricated platelet membrane-derived biomimetic nanobubbles for targeting acute ischemic brain tissues, which can be used for early intervention and serve as novel theranostic nanoformulations As a new emerging technology, biomimetic cell membrane coating provides a promising strategy for nanoparticles active targeting to ischemic tissues. Neutrophils and platelets have the ability to target inflammatory areas in response to chemokines et al. prepared a biosmart nanoparticle by encapsulating melatonin in the core and plasmids encoding VEGF in shell which was designed to protect cells by releasing melatonin to scavenge reactive oxygen species (ROS) at the acute stage and to secret VEGF for vascularization by sensing hypoxia at the chronic stage Accumulating evidence shows that miRNAs may play an essential role in cardiovascular diseases. Previous studies reported that miRNAs were responsible for angiogenesis, such as miR-126, which could block the expression of sprouty-related EVH1 domain-containing protein 1 (SPRED1) that was known to inhibit the angiogenic factor, VEGF de novo design strategy was proposed that artificial hybrid enzymes were created by in situ synthesis of Mn nanoparticles in recombinant human ferritin nanocages (FTn) and modified with a lipophilic cation, TPP, on the FTn surface for mitochondria targeting 2•- into H2O2 and subsequent conversion of H2O2 into H2O, which endowed protective roles in cardiac IR injury .The mechanisms of IR injury are complicate, but a well-characterized initiating factor is the burst of ROS from mitochondrial respiratory chain upon reperfusion. High concentration of ROS generation from mitochondria resulted in oxidative stress, loss of mitochondrial function and eventually death of cardiomyocytes. Therefore, development of mitochondria-targeting ROS scavenging nanoparticles may be a feasible strategy for the treatment of IR injury. A et al. found that MSC-derived exosomes significantly inhibited cardiomyocyte apoptosis and reduced the infarct size by inducing cardiomyocyte autophagy via targeting AMPK and Akt pathway 3O4 core and a silica shell modified with two types of antibodies which could specifically bind CD63 antigen on the exosomes and myosin-light-chain on the injured cardiomyocytes, respectively Exosomes are nano-sized membrane vesicles derived from a variety of cell types and can mediate cell-to-cell communications and convey immune-modulatory and pro-angiogenic signals. Exosomes released by MSCs contain various cargoes including proteins and miRNAs with the potential for angiogenesis and cardiac regeneration. Numerous studies have proved that MSC-derived exosome/extracellular vesicles are versatile delivery systems for the treatment of ischemic diseases. For example, Liu 2 decoration was designed, which exhibited natural infarct-homing ability in the pathological cardiac microenvironment and sustained release of paracrine factors to promote angiogenesis Figure 4).Mounting evidences suggest that stem cells exert their therapeutic effects primarily through paracrine mechanism Timely blood supply in the cerebral ischemic lesion can reverse tissue damage and inhibit neurological injury. Nanoparticles based angiogenesis gene delivery which could effectively avoid the risk of gene degradation has been developed for successful ischemic stroke therapy. As above mentioned, HIF-1α regulates the transcription of many pro-angiogenic genes. Prolyl hydroxylase 2 (PHD2) silencing alone was demonstrated to stabilize HIF-1α and increase the survival ability of endothelial progenitor cells (EPCs) in ischemic lesions et al. found that iron oxide nanoparticles themselves upregulated CXCR4 expression of MSCs by mediating HIF-1α expression, which improved MSC homing to injured area Insufficient secretion of neuroprotective factors and inadequate cells homing to the ischemic cerebrum make MSC-based therapy compromised in practical applications. For the first time, Huang 2 delivery to the ischemic cerebral tissues is of pivotal importance 2 after thrombolysis lead to serious impairment. Hemoglobin (Hb) that is a tetramer with four oxygen-binding sites has showed great potential as an oxygen reservoir in the application of regenerative medicine for oxygen supply. However, the short half-life limits the clinical application of Hb. Liposomal Hb could not only prolong the circulation time of Hb in vivo, but also deliver sufficient O2 to microcirculation where red cells seldom reach 3O4 nanosponge that could release oxygen and scavenge ROS was proposed. This strategy represents an attractive design to regulate the microenvironment of ischemic stroke.For patients who have microcirculation impairment, Oνβ1 specific binding ligand. In addition, smoothened agonist, an activator of sonic hedgehog signaling, was released in response to the acidic environment for angiogenesis promotion and neurological function recovery et al. conjugated RGD peptides (arginine-glycine-aspartic acid) with hyperbranched cationic polymer (DMAPA-Amyp) to prepare RGD-DMAPA-Amyp nanoparticles for HIF-1α plasmid delivery, which could target injured endothelial cells and accumulate in the ischemic cerebral tissues Figure 5).Currently, drug delivery nanoparticles for the treatment of ischemic stroke are less than satisfactory, mainly due to the presence of BBB and the short circulation time of nanoparticles. It has been reported that ischemia contributes to BBB opening, which is attributed to endothelium activation, leukocyte recruitment, cytokine production and edema formation The administration of MSCs has attracted great attention for the treatment of ischemic stroke, whereas the low delivery efficiency of MSCs to the stroke lesion limits their clinical application 2O3 magnetic nanoparticles (PAMNs). The PAMNs specifically targeted and accumulated at the ischemic tissues under an external static magnetic field gradient and produced NO in situ after tail vein administration, which promoted the revascularization and blood flow Endothelium-derived nitric oxide (NO), as a vessel-functional molecule, can regulate vasodilation, inhibit the aggregation of platelet and leukocytes and promote angiogenesis. Therefore, targeted delivery of NO to the ischemic lesion can recanalize the blocked microvascular. Based on this, a biomimetic nanoparticle was constructed by a platelet membrane encapsulated with L-arginine and γ-FePrevious study has demonstrated that conjugation of VEGF on gold NPs maintained VEGF bioactivity, promoted new blood vessels formation and increased blood perfusion which reached over 90% of normal tissues, whereas the same amount of free VEGF did not result in any significant improvements. In another instance, using the supramolecular self-assembly strategy, a peptide from insulin-like growth factor-1(IGF-1) folded into 3D supramolecular nanomaterials which exhibited a low dissociation constant to IGF-1 receptor and efficiently activated the IGF-1 downstream pathway. More importantly, the formation of supramolecular nanofibers showed superior bioactivity over native IGF-1 due to the enhanced biological stability 2O2 as core and PLGA as shell. Oxygen release was dependent on the diffusion of PVP/H2O2 complex through PLGA shell, and then H2O2 was turned into O2 by catalase embedded in gelation hydrogel. The duration of oxygen releasing lasted 4 weeks, which augmented survival of injured cardiac cells 2 may lead to excessive ROS formation since O2 cannot been released controllably in response to tissue oxygen concentration. Furthermore, Guan et al. developed microspheres with an oxygen responsive shell which contained 2-nitroimidazole whose degradation rate increased when the environmental oxygen level decreased. This property allowed the microspheres to release O2 when sensing hypoxic microenvironment. When co-delivery of the microspheres and MSCs to the mouse ischemic hindlimb, this system was capable of releasing O2 controllably and increased MSCs survival and their paracrine effects 2 to ischemic tissues to improve endogenous and exogenous cell survival, which can avoid cell death causing by excessive oxygen release.The establishment of angiogenesis typically takes days to weeks, which is largely unable to meet the requirement of revascularization of ischemic tissues, especially in the circumstance of acute ischemic events. Thus, there is an urgent need for developing oxygen-release system to elevate tissue oxygen level and improve cell survival. Recently, researchers developed an oxygen-release system by combination of hydrogel with microspheres based on a core-shell structure with PVP/HRecent studies showed that MSCs homing to ischemic tissues is mediated by interaction between stromal-derived factor (SDF) secreted by injured tissues and CXCR4 expressed on MSCs in vitro conventional culture, the secretome and membrane composition of MSCs which suffer inflammatory and hypoxic conditions in vivo in the infarct area were absolutely different. We recently constructed 3D MSCs spheroids to mimic the hypoxia microenvironment, and the differences of protein content were compared between 3D MSCs spheroids and 2D MSCs. Vascular regeneration-associated proteins were the topmost elevated proteins identified in 3D cell spheroids. Furthermore, a microparticle system by integration the secreted factors from 3D cell spheroids with cell membrane-derived coating was developed and showed superior revascularization effects in hindlimb ischemia model Unlike e.g., VEGF) tends to disequilibrate physiological states, leading to various proinflammatory diseases. In addition, the complex angiogenic factor cascades are currently mapped only incompletely. There are many efforts to develop pro-angiogenic drug-based therapies in recent years, but only a single pro-angiogenic drug has reached the market. Regranex that contains recombinant human PDGF-BB was approved for the treatment of diabetic foot ulcers e.g., embryogenesis), as well as pathological  states. In other words, modulation of hypoxic status in disease sites or delivery of hypoxia-mastering cocktail factors would be an ideal choice for inducing vascular regeneration in ischemic tissues.Therapeutic vascular regeneration is essential for ischemic diseases but remains a major challenge. Direct delivery of the factors  Detailed mechanism of enhanced vascular permeability of ischemic tissues. Several aspects are still unclear, such as size cutoff of nanoparticles and dynamic permeability changes after ischemia, which guide to design the desired nanomedicines and understand optimal therapeutic time window. ii) Choice of surface markers for active targeting. Currently, the available surface markers in ischemic tissues are limited, leading to the lack of design principles for active targeting of nanomedicines. Nanoparticles intravenously administered into body can be delivered to the disease regions via passive targeting and/or active targeting. Passive circulation and extravasation of NPs are dependent on their blood circulation time and the accumulation in specific disease regions as a result of certain pathological conditions. Although this rationale for the design of passive targeting NPs is well accepted, recent studies suggest that passive targeting is not always effective and reliable for nanoparticle delivery. It may be compromised by a number of biological barriers, such as kidney and lung epithelia, blood-brain barrier. Whereas, functionalized NPs with targeting ligands can enhance the effects of passive diffusion due to improving disease regions specificity while minimizing unwanted off target effects


A 25-year-old female who presented with stroke-like symptoms during sexual intercourse was found to have a patent foramen ovale (PFO). She was diagnosed with a cryptogenic transient ischemic attack (TIA) and underwent a successful catheter-based PFO closure. She had complete resolution of symptoms during both intercourse and physical activity. The septum between the right and left atriums of the embryological heart contains a window, referred to as the foramen ovale, that allows blood to shunt based on the physiologic needs of the fetus . While tWhile associations have been made between young adults with PFOs and cryptogenic strokes/transient ischemic attacks (TIAs), there is still insufficient data to draw causation between the two. This case describes a young female who experienced recurring stroke-like symptoms during sexual intercourse and subsequently underwent PFO closure.A 25-year-old, otherwise healthy female, presented to her primary care physician complaining of episodes of lightheadedness during sexual intercourse. She reported that these symptoms have always been present during sexual intercourse; however, the most recent episode, approximately five months prior, included dysarthria, numbness of bilateral upper extremities, bilateral vision loss, and unilateral facial drooping lasting approximately five minutes prior to resolution. She also endorsed similar episodes during intense physical activity. The patient denied any history of leg pain, leg swelling, or shortness of breath during any of the episodes or at presentation. The patient had no pertinent medical, surgical, family, or social history. There was no history of smoking or use of estrogen-containing birth control, and her body mass index was appropriate for her height and weight at 22.5. Vitals were all within normal range for her age. Physical examination was unremarkable including a cardiovascular examination that showed normal rate and sinus rhythm with no murmurs, rubs, or gallops and strong palpable distal pulses. Neurological examination revealed intact cranial nerves and no motor or sensory deficits. The patient was examined by cardiology one month later. The initial differential diagnosis included PFO, atrial septal defect (ASD), ventricular septal defect (VSD), vasovagal reflex, and complex migraine. On initial workup, laboratory samples including complete blood count (CBC), complete metabolic panel (CMP), and lipid profile were all within normal limits. Transthoracic echocardiogram (TTE) showed a small PFO with predominantly right to left shunting across the atrial septum and a possible small restrictive membranous VSD. As seen in Figure The patient underwent a percutaneous, transcatheter PFO closure approximately one month after the confirmative TEE. Intracardiac echocardiography confirmed the TEE findings of a modest-sized PFO that then easily crossed with a multipurpose catheter. The septum secundum was not thick and there was no atrial septal aneurysm, so a 25 mm Amplatz PFO occluder  was implanted with no residual shunt and normal left atrial pressure. The patient was started on daily 81 mg aspirin and 75 mg clopidogrel.At her follow-up appointment in the clinic, the patient reported heart palpitations two weeks post-procedure, so a 30-day event recorder was placed. No atrial fibrillation or pauses of three seconds or longer were noted, thus the recorder was discontinued. The patient reported no symptoms during sexual intercourse or physical activity two weeks post-procedure. As seen in Figure There are several studies describing young adult females with a PFO with stroke-like symptoms during sexual intercourse . These pThe question remains as to why this patient experienced classic stroke symptoms but had full resolution of symptoms and no remaining deficits. Gordy and Rowell reported that a lethal volume of air in an acute bolus for humans is theorized to be 3-5 ml/kg (300-500 ml of gas introduced at a rate of 100 ml/sec) but the rate of accumulation and patient position are contributors to lethality . This isMultiple studies have investigated a superior therapy for the prevention of recurrent cryptogenic strokes, including the RESPECT, Gore REDUCE, CLOSE, and DEFENSE-PFO trials. In a recent meta-analysis comparing PFO closure, anticoagulation, and antiplatelet therapy, patients under the age of 60 had a 64% lower risk of stroke recurrence when assigned to the PFO closure group versus the antithrombotic group . AdditioWhile studies suggest that PFO closure is superior to antithrombotic therapy in a subset of patients, further research is needed in patients with TIAs to look at recurrence, all-cause mortality, and risk versus benefit of procedural complications. The data is not as clear for patients who have experienced TIAs, mostly likely because of the nonspecific symptoms, lack of objective neuroimaging findings, and diagnostic nuances. The lack of pre-closure investigations, such as brain imaging and transcranial Doppler during intercourse, limits our ability to determine the true pathogenesis in this patient and others with similar stroke-like symptoms or TIAs. Future studies that include these tests and investigate mechanisms such as air embolisms may provide answers and lead to early detection and treatment to prevent recurrent symptoms.This case demonstrates a unique presentation of stroke-like symptoms during sexual intercourse in a 25-year-old female with a PFO. Further research on PFOs and associated pathological conditions is necessary to explore their contribution to cryptogenic strokes/TIAs. Identifying plausible mechanisms and underlying pathophysiology can help us better understand how to identify patients who are more susceptible to experiencing similar events and subsequently prevent patients from developing serious adverse effects.


Cerebellar structural and functional abnormalities underlie widespread deficits in clinical, cognitive, and motor functioning that are observed in schizophrenia. Consequently, the cerebellum is a promising target for novel schizophrenia treatments. Here we conducted an updated systematic review examining the literature on cerebellar stimulation efficacy and tolerability for mitigating symptoms of schizophrenia. We discuss the purported mechanisms of cerebellar stimulation, current methods for implementing stimulation, and future directions of cerebellar stimulation for intervention development with this population.Two independent authors identified 20 published studies  that describe the effects of cerebellar circuitry modulation in patients with schizophrenia or animal models of psychosis. Published studies up to October 11, 2022 were identified from a search within PubMed, Scopus, and PsycInfo.Most studies stimulating the cerebellum used transcranial magnetic stimulation or transcranial direct-current stimulation, specifically targeting the cerebellar vermis/midline. Accounting for levels of methodological rigor across studies, these studies detected post-cerebellar modulation in schizophrenia as indicated by the alleviation of certain clinical symptoms (mainly negative and depressive symptoms), as well as increased frontal-cerebellar connectivity and augmentation of canonical neuro-oscillations known to be abnormal in schizophrenia. In contrast to a prior review, we did not find consistent evidence for cognitive improvements following cerebellar modulation stimulation. Modern cerebellar stimulation methods appear tolerable for individuals with schizophrenia, with only mild and temporary side effects.Cerebellar stimulation is a promising intervention for individuals with schizophrenia that may be more relevant to some symptom domains than others. Initial results highlight the need for continued research using more methodologically rigorous designs, such as additional longitudinal and randomized controlled trials.https://www.crd.york.ac.uk/prospero/], identifier [CRD42022346667].[ The cerebellum was traditionally considered a primary driver of motor coordination ; howeverPrior to the current use of non-invasive neurostimulation methods to target the cerebellum, studies in the 1970–1980s used surgical methods to implant a cerebellar pacemaker in patients with schizophrenia –20. ThisRecent technological advances led to more effective and tolerable, non-invasive brain stimulation methods that can be safely applied to the cerebellum , 23. ConIn addition to magnetic stimulation approaches, electrical cerebellar stimulation methods have also been used, albeit not as commonly. Through the use of anodal or cathodal stimulation, tDCS can modulate cortical excitability of targeted neural circuits by either increasing or decreasing intrinsic neural firing . tDCS enPrevious brain stimulation studies in schizophrenia have typically targeted cortical regions, such as the frontal cortex and the motor cortex, in an effort to improve positive symptoms , 39, negvia cerebello-cerebral circuits , a class of GABAergic inhibitory neurons found in the superior cerebellum . PCs arevia a comparable system (N-methyl-D-aspartate receptors (NMDAR)  , 61. The (NMDAR) , 62. Bot (NMDAR) –68.Additionally, the effects of neurostimulation can extend throughout the brain, beyond the initial stimulation target. For instance, tDCS effects may influence activity in both the specific target region and multiple network systems by way of increasing/decreasing release of monoamine neurotransmitters, like dopamine, onto circuits that do not even involve the anodal stimulation site , 70. StuOver two decades ago, Andreasen et al.  called aAn initial systematic review reported on 10 studies  of cerebellar stimulation in schizophrenia . These sThis manuscript provides a systematic update regarding the effects of cerebellar stimulation in schizophrenia. We discuss the effects on clinical symptoms, cognition and behavior, functional brain networks and underlying neuro-oscillations, movement, and physiology. We also review the tolerability of this intervention method for individuals with schizophrenia. We close by discussing issues and technical considerations regarding implementation of cerebellar stimulation as well as recommendations and future directions.in situ injection), among animal models of schizophrenia, patients with schizophrenia-spectrum disorders, and individuals at risk of psychosis were included in the review. Articles were included if they included just an active stimulation arm comparing baseline to post-stimulation, or if they included a comparison of active cerebellar stimulation to a sham or active control condition. Unpublished papers and clinical trials were excluded from the systematic review. We systematically searched titles and abstracts using the following Boolean search terms: schizoph* AND cerebell* AND . References from all included papers as well as a previous systematic review (This systematic review was pre-registered on PROSPERO (CRD42022346667) and adheres to PRISMA guidelines . JPYH sec review  were alsQuality of included studies was classified based on Nathan and Gorman’s criteria  for ratiThe systematic literature search yielded a total of 1,510 published studies see , 2.Collapsed across included articles, this review included 283 patients with schizophrenia, 24 individuals scoring high on schizotypy scales, 9 rodents whose brains were manipulated to simulate schizophrenia-like deficits, and 28 healthy controls. Of the chronic schizophrenia studies, 3 studies recruited patients with moderate symptoms and 7 studies recruited treatment-resistant patients, as defined as patients whose symptoms were unsuccessfully treated through multiple courses of different antipsychotic medications.Researchers used a variety of study designs, with 7 studies being RCTs, 8 being open-label uncontrolled studies (1 pilot study), 4 case reports, and 1 preclinical study. Of the non-invasive brain stimulation studies, 9 were longitudinal, with the longest follow-up timepoint at 6 months (but the majority were under 6 weeks). Of all studies involving humans, 4 met criteria for Type 1 (RCTs with a sample ≥ 40), 3 for Type 2 (RCTs with a sample < 40), 3 for Type 3 (open-label uncontrolled studies), and 9 for Type 6 (descriptive and case studies). Note that study level criteria were not applied to the preclinical study because the criteria were based on human standards. Although the cerebellar pacemaker papers were technically open-label uncontrolled studies, they were categorized as Type 6 because of the use of clinical summaries as well as broad and non-specific treatment outcome categories. As can be seen in Most of the included studies used TMS (11 studies), with 6 studies using iTBS, specifically. Three studies used tDCS, 1 used tPCS, and 4 used cerebellar pacemakers . The preclinical study used delta-frequency optogenetic stimulation targeting the right lateral cerebellar nuclei. Of the non-invasive brain stimulation studies, 12 studies used repetitive magnetic pulses, and 10 studies included multi-session designs with 10-session designs most commonly used. Eleven studies stimulated the cerebellar midline/vermis.Cerebellar pacemaker studies were the pioneer studies that tested the effectiveness of cerebellar stimulation on alleviating symptoms and improving functioning in schizophrenia. These treatment-resistant, small sample studies showed modest improvements in clinical symptoms and functioning  at different follow-up periods –20; howeOf the studies utilizing non-invasive brain stimulation, 12 examined the effects of cerebellar stimulation on clinical symptoms. The most consistently examined clinical domain was psychotic symptoms, with all 12 studies including measures of psychotic symptoms; 10 studies specifically used the total and/or the positive, negative, and general psychopathology subscales from the Positive and Negative Syndrome Scale (PANSS) . Three oSeven studies examined the impact of cerebellar stimulation on total symptom scores, which are a combination of negative, positive, and general pscyhopathology symptoms. One study found a specific effect of active rTMS in reducing total symptoms, but this effect was not significant when accounting for baseline total symptom level . SeveralNs ranging 11–64) with active/sham rTMS stimulation protocols  observed significant Stimulation X Time interactions for PANSS negative symptoms  and one pilot study found improvement on clinical global impression post-iTBS or tDCS, respectively (N = 8) did not find a change in clinical global impression following iTBS , though we note this sample was quite small (N = 11).Six studies examined the effects of cerebellar stimulation on underlying brain dynamics, with two studies utilizing resting-state functional connectivity and four studies utilizing electroencephalography (EEG) to derive outcome variables. There is a well-established literature documenting aberrant connectivity between the cerebellum and prefrontal cortex in schizophrenia . In a laUnderlying neuro-oscillations measured with EEG can be abnormal in schizophrenia, such as frequencies associated with perception, memory, and synaptic plasticity, including theta and gamma –99. ThetThree studies examined the effects of cerebellar stimulation on movement-related symptoms. Two RCTs found no significant effect of iTBS. In one study, individuals with schizophrenia showed decreased extrapyramidal symptoms and ataxia at 6-week follow-up irrespective of their treatment condition . In the Two studies examined the effects of cerebellar stimulation on physiology . One RCT found no effect of iTBS; individuals with schizophrenia showed decreased diastolic blood pressure at 6-week follow-up irrespective of their treatment condition . An openAs described earlier, pioneer studies using invasive cerebellar stimulation methods –20 had pWhen solely focusing on the 7 RCTs that examined cerebellar stimulation in schizophrenia, results remain largely the same as when all studies are included because the results from RCTs (Types 1 and 2) in this systematic review took precedence over less rigorous study designs (Types 3 and 6).This updated systematic review covers available evidence of cerebellar stimulation effectiveness in treating different symptoms of schizophrenia and influencing underlying neural systems that are deficient in schizophrenia. The number of included studies has more than doubled since the last systematic review , and mulOver 80% of articles examined whether cerebellar stimulation could alleviate clinical symptoms of schizophrenia. There was some evidence that cerebellar stimulation reduced total psychotic symptoms . However, it is unclear whether this reduction was driven by a more specific reduction in negative symptoms. Negative symptoms are thought to account for much of the long-term morbidity, functional impairments, and poor quality of life in schizophrenia, and as such remain a critical unmet need in schizophrenia treatment . CerebelSchizophrenia and depression are highly comorbid disorders , with boSurprisingly, cerebellar stimulation did not improve cognition in people with schizophrenia. This contrasts with research in non-psychiatric groups as well as non-psychotic cerebellum-involved disorders, in which participants showed significant gains in learning post-cerebellar stimulation , 108. WhFew studies have looked at whether cerebellar stimulation can impact specific behavioral changes. Interesting findings in rodents showed changes on an interval timing task that captures one’s ability to maintain various temporal intervals in working memory ; this stvia the cerebellum could be an approach to improving symptoms in schizophrenia. Studies examining EEG-related oscillations found that individuals with schizophrenia had a more normal pattern of increased theta oscillatory power in the midfrontal region , most studies have followed standard parameters from cortical stimulation studies . PrelimiAnother technical issue to consider when conducting cerebellar stimulation RCTs is selection of the sham condition. There is great variability in sham methods employed in the field . Selection of the sham condition can lead to differential biological effects beyond the intended transient sensations, which in turn affect the results . For shaA major advancement in this field (that is currently underway) is the implementation of RCTs to evaluate cerebellar stimulation in schizophrenia , 13. 57%It has also been argued that research linking clinical symptoms to neurobiological measures is hampered by research design obstacles, many of which were present across these studies . NotablyLongitudinal designs that extend beyond 6 weeks can help clarify the longevity of effects and whether additional doses/boosters are needed. More nuanced longitudinal studies could also help to clarify whether there are individual plateaus in treatment effects, i.e., the subject-specific point after which there are diminishing returns. Available studies varied widely in terms of when they assessed treatment effects. While some studies assessed change throughout the stimulation period, others only compared pre- and post-completion timepoints.Additional research is needed to understand who will most benefit from cerebellar stimulation . Many stAnother future direction is to combine cerebellar stimulation with multiple neuroimaging modalities  and behavioral tasks to drill down on the underlying circuits impacted by cerebellar stimulation . That isA caveat regarding the results of this systematic review is the potential for publication bias, especially since many of the older included studies were small open-label or case studies. Although a systematic review was conducted on multiple databases for published articles, studies finding null results might not have been published due to rejection based on small sample size or because the authors did not attempt to publish the results. As studies in the field shift to larger and more rigorous RCTs or longitudinal designs, which can provide more power for detecting effects and can reduce the probability of a Type II error that is more prevalent in small sample studies, the likelihood of publication of null results becomes greater.Taken together, cerebellar stimulation shows potential for alleviating negative and depressive symptoms in people with schizophrenia. The mechanism of action underlying cerebellar stimulation may be through modulation of underlying brain systems and oscillatory dynamics, consistent with previous suppositions that targeting the cerebellum can have widespread impact due to its role in distributed cerebellar-cerebral networks. Advancements in cerebellar stimulation have great treatment potential for schizophrenia, although improved standardization across studies is needed to establish the best practices for implementing these approaches and to identify the specific clinical features of schizophrenia that are most responsive to cerebellar stimulation.The original contributions presented in this study are included in the article, further inquiries can be directed to the corresponding author.JH was responsible for compiling the initial set of studies for further evaluation. JH and SA were responsible for selecting the final set of included studies and for extracting data from these studies and drafted the initial manuscript. All authors were responsible for the study concept and design, critically reviewed the manuscript, and approved the final version for publication.


The airway epithelium of children with asthma is characterized by aberrant repair that may be therapeutically modifiable. The development of epithelial-targeting therapeutics that enhance airway repair could provide a novel treatment avenue for childhood asthma. Drug discovery efforts utilizing high-throughput live cell imaging of patient-derived airway epithelial culture-based wound repair assays can be used to identify compounds that modulate airway repair in childhood asthma. Manual cell tracking has been used to determine cell trajectories and wound closure rates, but is time consuming, subject to bias, and infeasible for high-throughput experiments. We therefore developed software, EPIC, that automatically tracks low-resolution low-framerate cells using artificial intelligence, analyzes high-throughput drug screening experiments and produces multiple wound repair metrics and publication-ready figures. Additionally, unlike available cell trackers that perform cell segmentation, EPIC tracks cells using bounding boxes and thus has simpler and faster training data generation requirements for researchers working with other cell types. EPIC outperformed publicly available software in our wound repair datasets by achieving human-level cell tracking accuracy in a fraction of the time. We also showed that EPIC is not limited to airway epithelial repair for children with asthma but can be applied in other cellular contexts by outperforming the same software in the Cell Tracking with Mitosis Detection Challenge (CTMC) dataset. The CTMC is the only established cell tracking benchmark dataset that is designed for cell trackers utilizing bounding boxes. We expect our open-source and easy-to-use software to enable high-throughput drug screening targeting airway epithelial repair for children with asthma.  Frequent exposure to pathogens, allergens and pollutants results in damage of the epithelial cell layer lining the airways . FollowiVarious in vitro assays can be used to study wound repair mechanisms  and screManual cell tracking is laborious, slow, subject to bias ,12 and cAutomated cell trackers generally operate by first outlining the exact shapes of, or segmenting, cells in images before tracking their movements across multiple frames . Cell seImportantly, most automated trackers rely on algorithms that do not presuppose that image sequences have low resolution  and lowhttps://github.com/AlphonsG/EPIC-BBox-Cell-Tracking, accessed on 9 February 2022). EPIC uses state-of-the-art AI-based Vision Transformers [Here, we present EPIC, a fully automated cell tracking software solution that overcomes all the previously mentioned limitations  in culture media lacking epidermal growth factor. We wounded confluent monolayer cultures using the IncuCyte® 96-well WoundMaker Tool (Essen Bioscience) [® ZOOM (Essen Bioscience) [We generated a wound repair dataset under high-throughput drug screening experimental conditions. We obtained human telomerase reverse transcriptase modified airway epithelial cells (NuLi-1)  from thescience) . We thenscience) . We refeUnlike the long-existing Cell Tracking Challenge , the recWe trained a state-of-the-art AI system based on Vision Transformers to detect low-resolution airway epithelial cell nuclei and whole cells in our wound repair dataset and the CTMC dataset, respectively, using bounding boxes. We then developed a custom algorithm capable of tracking cells at low resolution and low framerate. The algorithm extracts two appearance and four motion features from cells detected in every frame of an image sequence. The extracted features are used to link cells with the same identity across frames with a custom multi-stage tracklet association and tracklet cleaving strategy based on combinatorial optimization ,57,58. WWe compared EPIC’s cell tracking performance in our wound repair dataset to manual cell tracking, as previously described . We alsoOur wound repair dataset was 380% lower resolution (1.33 µm/px) than the CTMC dataset (0.35 µm/px). Furthermore, all cells in our dataset were classified as small objects (<32 by 32 bounding box area) . The CTMThe training of the cell detection model was completed in ~30 min. The model achieved average precision and recall values of 87% and 91%, respectively. These metrics can be interpreted as the model correctly detecting ~90% of cells in each image. As shown in In contrast, DeepSORT failed to track any cells from the 1st to the 22nd frame without fragmentation in the same experiments, and with no cells tracked in the initial frames, no leading edge cell tracks could be defined and hence sampled for wound repair analysis .Viterbi had the most variable cell tracking performance in the same experiments, tracking anywhere from 0 to 2726 cells from the 1st to the 22nd frame without fragmentation depending on the experiment . We noteWound Repair AnalysisThe sampled leading edge cell tracks that were generated by EPIC were largely similar to 20 randomly selected and manually tracked leading edge cells in the corresponding experiments, while sampled leading edge cell tracks generated by Viterbi were largely dissimilar to those manual cell tracks . Unlike We used the manually and automatically generated leading edge cell tracks sampled from the nine total tested delayed, control and accelerated experiments to compute six cell migration metrics: Euclidean distance, accumulated distance, velocity, directionality, Y-forward migration index and end point angle. Cell migration metrics produced by manual cell tracking and EPIC were similar and both were different from metrics produced by Viterbi . The celStatistical comparison of the cell migration metrics produced by EPIC cell tracks indicated that there were no statistically significant differences to those produced by manual cell tracking for all metrics . On the We found that wound repair analysis outcomes of untreated and drug-treated cells according to EPIC were equivalent to those of the current gold standard method, manual cell tracking, while generating at least 20 leading edge cell tracks across the 22 image frames without any user labor required. In contrast, Viterbi did not consistently track at least 20 leading edge cells across 22 frames. Additionally, Viterbi produced wound repair outcomes contradicting the expectations for untreated and drug-treated cells, and many of Viterbi cell tracks were visually incorrect. For instance, closer inspection revealed that, unlike EPIC , many ofEPIC automatically identified the leading edges of the wound in all experiments . By visuEPIC detected and tracked thousands of cells in all the nine experiments in ~20 min . In contThe tracking of hundreds of small objects in an image sequence is a challenging task outside the intended use cases of most existing trackers. Accordingly, EPIC’s efficient design and performance enhancements, such as advanced multicore processing, resulted in magnitudes faster processing times than Viterbi and DeepSORT. We anticipate Viterbi and DeepSORT’s long processing times to render these methods infeasible for use in high-throughput drug screening pipelines involving thousands of experiments. Although manual cell tracking is infeasible for high-throughput drug screening, the 20 cells from each delayed or control experiment were manually tracked within 10 min. Cells from each accelerated experiment were manually tracked within 30 min due to increased difficulty tracking fast-moving cells at the low framerate. The total manual tracking time for all experiments (180 cells) was 2.5 h.EPIC outperformed Viterbi and DeepSORT in cell tracking accuracy on the CTMC dataset . TherefoWe utilized object orientated software design principles, such as the Factory Method creational software design pattern, to allow developers to easily substitute custom object detectors and even tracking algorithms into EPIC to better suit other use cases, allowing for a highly flexible system. For researchers using EPIC ‘out-of-the-box’, it is a cross platform application that is simple to use through a command line or graphical user interface. Through the available commands, users can perform object detection, tracking and analysis of time lapse images in common formats, such as TIFF and JPEG. EPIC also automatically performs tracking and migration analyses of all cells in wound repair experiments. Generated cell tracks can be exported in multiple formats, such as ImageJ Manual Tracking File  and MOTCEPIC automatically tracked unstained cells (including drug-treated and untreated control) as accurately as manual cell tracking in a challenging low-resolution, low-framerate and high cell density dataset at higher volume and speed. This is unlike tested publicly available automated trackers, which underperformed on such a challenging dataset and hence cannot be reliably used for high-throughput wound repair analyses. EPIC also outperformed the same trackers on the diverse and challenging CTMC dataset, which includes 14 different cell lines, reinforcing that EPIC is not limited to airway epithelial repair for children with asthma but can be applied in other cellular contexts. EPIC tracks cells detected using state-of-the-art AI-based Vision Transformers and bounding boxes with a custom tracking algorithm. This results in highly accurate cell tracking with decreased labor and time necessary for training data generation compared to widely used segmentation-based approaches. We expect our open-source and easy-to-use software to enable high-throughput drug screening targeting airway epithelial repair for children with asthma.


These effects limit access to artifact-free optical measurements during peripheral neurosurgery to only a portion of the exposed nerve without further modification to the procedure.Modern optical volumetric imaging modalities, such as optical coherence tomography (OCT), provide enormous information about the structure, function, and physiology of living tissue. Although optical imaging achieves lateral resolution on the order of the wavelength of light used, and OCT achieves axial resolution on a similar micron scale, tissue optical properties, particularly high scattering and absorption, limit light penetration to only a few millimeters. In addition, in vivo OCT imaging during peripheral neurosurgery in small and large animals by increasing the amount of visualized nerve volume as well as suppressing motion of the imaged area.We aim to improve We designed a nerve holder with embedded mirror prisms for peripheral nerve volumetric imaging as well as a specific beam steering strategy to acquire prism and direct view volumes in one session with minimal motion artifacts.The axially imaged volumes from mirror prisms increased the OCT signal intensity by We demonstrated improved OCT imaging during neurosurgery in small and large animals by combining the use of a prism nerve holder with a specifically designed beam scanning protocol. Our strategy can be applied to existing OCT imaging systems with minimal hardware modification, increasing the nerve tissue volume visualized. Enhanced imaging depth techniques may lead to a greater adoption of structural and functional optical biomarkers in preclinical and clinical medicine. For many applications, such as retinal imaging in ophthalmology, light penetration depth is adequate to glean sufficient information for clinical decision-making. However, as OCT expands to new applications in opaque tissues, light scattering and absorption can more adversely limit its utility. One such area is peripheral nerve imaging, where optical imaging modalities have been applied to collect important information about peripheral nervous system (PNS) conditions and the interaction of devices to relieve those conditions .–,in vivo volumetric structural and functional information.,in vivo peripheral nerve examination.,Linear imaging modalities, such as confocal microscopy and optical coherence tomography (OCT), provide depth-resolved capability for Typical OCT images of small animal peripheral nerve  obtained with a 1310-nm swept-source system are unable to resolve features in the bottom half of the nerve . For larA number of techniques have been proposed to increase OCT imaging quality by extending the optical depth-of-field, including beam shaping to produce Bessel,,The idea of using a mirror prism for illumination and detection in the axial plane was first demonstrated in confocal microscopyin vivo PS-OCT image acquisition techniques, and potential clinical use for ancillary real-time optical imaging during PNS device implantation in humans. First, we imaged tissue-mimicking phantoms in the nerve holders to verify and optimize performance. OCT subvolumes from side views appearing in-focus ensured the correct choice of size and material of the mirror prisms in small and large animal nerve holders. Then we performed acute PS-OCT imaging of rat sciatic and minipig vagus nerves to characterize and validate in vivo performance. In this paper, we demonstrate the suppression of motion during in vivo imaging as well as the acquisition of complementary volumetric information through side prism imaging. The platform enhances the information collected during optical imaging of peripheral nerves for a variety of emerging neuromodulation applications.Our custom nerve holder design was motivated by several factors, including application to both small and large animal studies, strategies for their use in a surgical environment, consideration of 22.1,in vivo imaging of sciatic and vagus peripheral nerves in small and large animals and configured for field portability for easy transport between surgical suites or between the laboratory and surgical suite. The optical head is mounted on a S21 Zeiss surgical microscope arm and the OCT processing engine, system computer, and other instrumentation were placed on a mobile cart  with optical breadboard . The optical head is tethered to the main OCT engine with two sets of electric cables to control galvanometer scanners and the sample path fiber optic cable.For testing the prism nerve holder, we used a swept-source  PS-OCT system that is more fully described elsewhere.The optical head and OCT engine are configured so that multiple low-NA objectives  can be swapped easily. Most often, the system was equipped with the LSM54 objective which provides a 64-mm working distance, especially to image the nerve in the deep surgical pocket of the minipig neck. The 3-mm beam diameter after the collimator provides near-diffraction limited ,PS-OCT images are acquired as follows. The system images at 50,000 A-lines per second. The polarization of the A-lines is modulated between two orthogonal states such that adjacent A-lines are always orthogonal. Each A-line generates four interferograms, two of which describe the signal in a first polarization state and the remaining two in a second orthogonal polarization state. The two interferograms within each polarization state are fed to a balanced detector. This creates two voltage signals (one from each polarization state) that are digitized by a two-channel DAQ board  at 200 MHz synchronized to an external clock generator, sampling each interferogram with 3072 points. During acquisition, custom software writes sampled interferogram data into memory and displays live previews of the structural and angiographic cross-sectional OCT images. The system uses a custom scanning protocol: one volume consists of 1184 B-scans; each B-scan is reconstructed from 1184 lines divided into 4 backstitched segments of 296 lines.After image acquisition, each binary file, which contained the sampled interferograms, is processed by graphics processing unit (GPU)-accelerated  Python software2.2Peripheral nerve holders were used to stabilize the nerve from respiratory, cardiac, and general animal motion during imaging, which can corrupt OCT images, especially OCTA images, where even pixel-scale motion of a few microns will add speckle or phase noise to the images. In our previous studies, we demonstrated significant improvement in OCTA imaging when using a nerve holder mounted to the animal stage.First, the additional optical distance traveled for the side reflected beams means that the distance to the tissue will not match the objective focal length and the tissue will be out of focus, even for a long depth-of-focus objective. This would require inefficient acquisition of two volumes, one focused on the top of the tissue and a second focused on the side(s) after refocus . We solved this issue using back-surface reflecting prisms so that the OCT beam passes through the prism glass, extending the focal distance of the side reflected beams to match that of the top directed beam. In this case , the reqSeveral back-surface mirrored prisms with different refractive index glasses were tested to determine the best focus match without significant perturbation in the PS-OCT signal. Back-surface Al-coated mirrored prisms made of N-SF11 glass with AR-coated leg surfaces  were chosen.Second, the nerve holder yields a difference in apparent axial image position for the side regions corresponding to beams reflected off the prisms compared to the center region corresponding to the beam directly back-reflected from the nerve due to extended optical path length. Because all OCT systems experience some roll off in signal intensity with axial position, if the difference in the apparent axial positions is large, there can be a significant difference in the OCT signal intensity for the prism-reflected and direct back-reflected regions of the image. However, because the coherence range gate (CG) on our PS-OCT system is electronically controlled, it is possible to compensate this in the software and move the CG during a volume acquisition. Inherent timing jitter means the CG can only be adjusted slowly, and this adjustment is best done by switching the fast and slow axis so that the scanning is done along the prism regions first (CG2), then along the center region (CG1), and then adjusted back to CG2 for the other prism.Third, the design minimized specular reflections coupled back into the OCT signal by adding a small angular tilt to 3D-printed based on which the back-surface coated prisms were mounted and using antireflective coating on the prisms. As mentioned above, the optical head was also fit with a kinematic stage for moving the nerve axially into the focal plane and a tip-tilt to account for any angle in the nerve within the holder. The design considerations provide optimal conditions for peripheral nerve imaging in small and large animals.2.3in vivo within the size constraint set by the surgical pocket [CAD modeling  and additive manufacturing technology allowed us to rapidly design and fabricate several compact nerve holder bases for imaging tissue-mimicking phantoms, and small and large animal peripheral nerves l pocket . The desEither Objet260 Connex3  or Form 3  printers were used to fabricate nerve holders. Both printers provided sub-Cylindrical Nd magnets (diameter 1.2 mm and height 1 mm) were glued into the magnet cavities with the correct polarity according to the respective magnetic rod of the optical head. The final assembled nerve holder magnetically attached to optical head on the Zeiss S21 surgical microscope arm is shown in in vivo nerve tissue, the mechanical rigidity of a fixed nerve or a phantom preserves the circular shape in cross section during imaging. This leads to an increased difference in the focal length between the prism image and direct image. Therefore, we manufactured versions of the small and large animal nerve holders with larger prisms (5 and 10 mm) to account for increased focal lengths during phantom bench-top imaging.The different versions of the nerve holder allow maximum flexibility for various potential applications. The magnetic prism nerve holders for small animals , top row2.4,The prism nerve holder allows the OCT beam to probe nerve regions not normally accessible with direct top-down beam delivery. To aid nerve holder optimization and demonstrate initial operation and performance with a controlled target, we imaged tissue-mimicking phantoms with 1.2- and 3.5-mm cross-sectional diameters. Both phantoms were constructed from polydimethylsiloxane (PDMS) silicone, a commonly used phantom material.in vivo experiments, we also imaged ex vivo nerve samples (not presented in this paper). Finally, imaging small (rat) and large (minipig) animals using magnetic prism nerve holders demonstrated full in vivo operation and performance.In preparation for 2.5Both rat and minipig surgical procedures were approved by the Institutional Animal Care and Use Committee at the U.S. Food and Drug Administration, White Oak Campus.Rattus norvegicus, female, The surgical procedure to expose the rat sciatic nerve was similar to one previously reported by Vasudevan et al.Sus scrofa domesticus, female, Healthy Yucatan minipigs , myelin sheath (Anti-Myelin P0), and nuclei . Immunohistochemistry (IHC) images were obtained on confocal microscope  using 33.1Tests with small (1.2 mm) and large (3.5 mm) tissue-mimicking phantoms demonstrated the increased light penetration without the need to mechanically adjust the imaging depth and verified that the geometrical parameters of the fabricated prism nerve holders are suitable for small and large animal peripheral nerve OCT imaging. Complementary information from the prism views provided fine structural details which were not accessible due to the limited penetration depth and highly scattering objects overshadowing structures below. However, functional OCT imaging modalities (OCTA and PS-OCT) require the presence of moving scatterers and birefringence,3.2en face projection [en face projections clearly demonstrate that images obtained from prism regions contain views of vascular and fascicular structure from the bottom half of the nerve that are not visible on direct view. Isometric projection of volume rendering of BwOA [We used the double-sided magnetic prism nerve holder to demonstrate improved OCT imaging in rat sciatic nerve . The imaojection  of the pojection . Large bojection . BwOA imojection  provides of BwOA  provides3.3in vivo from a minipig with the prism nerve holder using our PS-OCT system. Each image represents five averaged adjacent projections. Although fascicles can be seen on structural OCT [ural OCT  direct nural OCT , where tural OCT  demonstrEn face OCTA projections [en face projection due to apparent difference in imaging depth, those blood vessels are visible in the cross-sectional images. Additional noise mitigation and motion suppression is achieved with a scanning protocol acquiring B-scans along the nerve. jections  show a v4ex vivo nerve fascicle tracingin vivo surgical applications. Currently, x-ray and computed tomography are routinely used to assess the implantation of stimulation electrodes postoperatively;In vivo results in mouse brain demonstrated up to 1.2 mm penetration depth. However, the light scattering in peripheral nerves is higher than in brain, and there is limited availability of components for light delivery and detection at mid-infrared wavelengths. This currently creates a challenge for accessing PS-OCT derived biomarkers and translation of longer wavelength OCT systems for surgical applications.Deep tissue penetration and side visualization of peripheral nerves in living animals provides significant additional structural information—including nerve fascicle and blood vessel traces—to aid a variety of neurostimulation and neuromodulation applications. Although recent reports of Our PS-OCT system with 1300-nm central wavelength provides a compromise between penetration depth  and side (prism) views, initially subsequent to acquisition but eventually in real time with the aid of parallel processing hardware  or machine learning algorithms. We made initial progress toward this goal with a software algorithm that uses a geometric transformation between landmarks manually chosen in one B-scan between top-down and side views, which is then applied with alpha blending to all B-scan in a set of multimodal  volumes. The merging algorithm works reasonably well on small animal nerves; however, large animal nerves present larger distortions due to refractive index differences between air and tissue.Anecdotally, our method of encoding tissue optical axis orientation with birefringence into multicolor images shows that some of the fascicles exhibit different optic axis orientation values. This can be observed in the bottom panels of The deep tissue imaging strategy presented here provides complementary information on PS-OCT derived imaging modalities using side views of a tissue target. The current prism nerve holder implementation does not disrupt the standard PS-OCT imaging path, does not require extensive hardware modifications,5in vivo rat sciatic nerves, and in vivo minipig vagus nerves. The prism nerve holder will improve OCT imaging of peripheral nerves and enhance the development of imaging-based biomarkers to prevent nerve injury during the treatment of various neurological conditions and other neuromodulation applications.We developed a prism-based peripheral nerve holder, imaging protocol, and postprocessing merged visualization software to provide enhanced penetration for PS-OCT imaging. We demonstrated the successful application of this approach on tissue-mimicking phantoms,


Mitochondrial DNA is primarily maternally inherited in most animals and evolves about 10 times faster than biparentally inherited nuclear DNA. Mitochondrial dysfunction (mt-dys) arises when interactions between the co-evolving mitochondrial and nuclear genomes are perturbed in essential processes like oxidative phosphorylation (OXPHOS). Over time mt-dys can lead to mitochondrial diseases (mt-diseases), which are surprisingly prevalent and include common diseases such as Alzheimer’s, Parkinson’s, and diabetes. Unfortunately, the strong impact that intraspecific mitochondrial and nuclear genetic variation has on mt-disease complicates its study and the development of effective treatments. Animal models have advanced our understanding of mt-disease but their relevance to human conditions is often limited by their relatively low nuclear genetic diversity. Many traditional laboratory models also typically have a single mitochondrial haplotype (mitotype), in stark contrast to over 5,000 mitotypes in humans worldwide. The threespine stickleback fish has an evolutionary history that has made it a favorable evolutionary mutant model (EMM) for studying mito-nuclear interactions and possibly mt-diseases. EMMs are species with naturally evolved states that mimic maladaptive human diseases. In threespine stickleback, a period of isolation followed by introgression of the mitochondrial genome from a sister species resulted in the maintenance of two distinct mitochondrial haplotypes which continue to segregate within many populations of wild stickleback. The existence of two mitogenomes segregating in numerous genetically diverse populations provides a unique system for exploring complex mito-nuclear dynamics. Here we provide the first complete coding region analysis of the two threespine stickleback mitotypes, whose mitogenomic divergence exceeds that of other mammalian models for mitochondrial disease and even that between ancient and modern humans. We find that divergence is not uniform across the mitogenome, but primarily impacts protein coding genes, and significantly impacts proteins in Complex I of OXPHOS. The full characterization of these highly divergent intraspecific mitotypes provides a foundation for the development of threespine stickleback as an EMM for mito-nuclear interactions. Typical metazoan mitochondrial genomes (mitogenomes) are small circular genomes encoding 13 proteins, two rRNAs and 22 tRNAs. Mitogenomes are overwhelmingly maternally inherited in most animals in contrast to the biparentally inherited nuclear genome , and theMt-dys and resultant mt-diseases are surprisingly prevalent in humans. Primary mitochondrial diseases (PMD), are defined as diseases caused by a mutation in a protein comprising a subunit of OXPHOS, including diseases such as Leigh’s Syndrome, Pearson Syndrome, and Barth Syndrome. The definition of mt-disease has further expanded to include secondary mitochondrial diseases (SMD). These include any disease exhibiting mt-dys as a symptom, such as Parkinson’s Disease, Alzheimer’s Disease, diabetes, cancer, muscular dystrophy, and amyotrophic lateral sclerosis (ALS) . The ideClinical studies have been extremely valuable in identifying disease-causing genetic variants , and modGasterosteus aculeatus) have an evolutionary history that makes them well-suited EMMs for mito-nuclear interactions  mitotype that had accumulated many mutations , modern human (Homo sapiens), and ancient human  mitogenomes .We assembled mitogenomes for 34 threespine stickleback to be compared to the reference genome  from Ensembl; total n = 36 mitogenomes. For population data, we extracted mitochondrial sequences from genomic data on the Sequence Read Archive (SRA) for six TNP fish from the Akkeshi River System in Japan and six ENA fish from Limfjord, Denmark. Accession numbers are summarized in ). When possible, we standardized DNA to 200 ng and pooled fragments for each sample prior to library preparation. We sheared the pooled amplicons to a range of 300–400 base pairs (bp) using sonication . Libraries were prepared from these fragments using end repair via NEB Quick blunting reactions, followed by addition of A-overhangs, T4 ligation to uniquely barcoded Illumina-compatible adapters, and total library amplification. We then multiplexed libraries and sequenced on the HiSeq4000 in the UO Genomics and Cell Characterization Core Facility (GC3F) generating paired-end 150 bp reads (We isolated DNA from somatic tissue using standard methods and amplified the mitogenome from these genomic templates via PCR. To do so we designed five primer pairs  to amplibp reads .https://www.geneious.com) using MUSCLE. We then trimmed the multiple alignment to the limits of the coding region to exclude the partially assembled control region. Therefore, we included annotated 22 tRNAs, two rRNAs, and 13 coding genes based on the reference annotation from Ensembl. Because tRNAs were not specifically annotated in the reference, we confirmed the identity of each tRNA using tRNAscan-SE v2.0 (https://www.geneious.com) via MUSCLE and annotated genes based on reference annotations from Ensembl.We aligned demultiplexed 150 bp reads to the Bear Paw TNP reference mitogenome from Ensembl using the Burrows-Wheeler Aligner (BWA)  and filt-SE v2.0 . For exthttps://www.geneious.com) and calculated population genetic statistics using DnaSP v6   in order to assess divergence among and within populations, as well as between ENA and TNP mitotypes. These included six genomes from a single population in Denmark (ENA), six genomes from a single population in Japan (TNP), and 22 individuals from different populations in North America (ENA). To standardize comparisons to other models of mitochondrial divergence, we trimmed the assemblies to exclude the highly variable control region and included the full suite of 13 protein coding, two rRNA, and 22 tRNA genes.We used maximum-likelihood phylogenetic analyses to assess divergence. We found that the majority of mitogenomic divergence was between mitotypes irrespective of geographic origin of population, as indicated by a clear phylogenetic clustering of all ENA mitotype fish to the exclusion of TNP fish with branch lengths between mitotypes greatly exceeding those separating other groups . We addiG. aculeatus would have undergone a population expansion at the end of the glacial maximum once they rejoined the Pacific Ocean populations  and ancient humans . These results confirm that what we document in the single stickleback species is an unusual amount of mitotype divergence, as it exceeds that of mitogenomic divergence between subspecies.We identified rates of intraspecific mitogenomic divergence between threespine stickleback mitotypes that exceeded that of previously identified notable intersubspecific mitogenomic divergence for two sets of subspecies pairs . We compthe taxa . We alsoIn several populations we identified fish that had polymorphic nucleotide sites in their mitogenome assemblies, indicating either paternal leakage of mitochondria or spontaneous mutations that led to more than one mitochondrial haplotype within single individuals. Such a condition is called mitochondrial heteroplasmy . In all trna-cys exhibited below genome-wide average sequence identity and the protein-coding gene mt-coII had above average identity. For protein-coding genes there was no correlation between gene size and average between mitotype percent sequence identity    through comparisons of North American ENA fish to the North American TNP reference genome , we foun = 0.53) ; Table 2 = 0.36) ; Table 3identity . Analysiidentity  includin element .We observed the highest rates of divergence in proteins involved in Complex I of OXPHOS comprised of ND1, ND2, ND3, ND4, ND4L, ND5, and ND6  suggestiWe found evidence of strong purifying selection in some protein-coding genes when we combined all TNP samples from Japan and North America to compare with the North American ENA sample. Using a McDonald-Kreitman 2x2 test for selection , we asseM m musculus and M.m domesticus  throughout the Northern Hemisphere. As such, there has not been enough time for the G. nipponicus TNP-mitogenome to replace the endogenous ENA-mitogenome  rather than among populations or within individuals. Second, the observed level of divergence also exceeds that of existing outbred models with whole mitogenome assemblies including These strengths are extremely valuable in addressing questions concerning mito-nuclear dynamics in the context of environmental and genetic heterogeneity. Fortunately, while threespine stickleback are a promising EMM due to their repeated mitotype admixture in nature, they are also amenable to laboratory studies. As in zebrafish, developmental and physiological studies are facilitated by external fertilization in stickleback . Threespmt-nd6 being a clear outlier for elevated levels of nucleotide divergence. Importantly, we discovered that much of the protein divergence is in those functioning early in OXPHOS in Complex I, even though Complex I proteins are spread throughout the mitogenome. More work is needed to understand what, if any, are the physiological consequences that exist because of divergence in Complex I proteins within populations. However, these findings are suggestive and intriguing, as Complex I is the primary producer of ROS, which, if not cleared from mitochondria by antioxidants and other proteins in Complex I, can lead to mt-dys and cellular damage (mt-cytb (mt-cytb was a serendipitous choice as a representative of average mitotype divergence, but it missed the more extreme divergence identified in other areas of the mitogenome. We propose that full mitogenome sequencing is therefore required for the advancement of EMMs for mito-nuclear interactions.Another strength of stickleback as an EMM is the heterogeneity of divergence across the stickleback mitogenome. We observed divergence primarily in coding genes irrespective of their physical location in the mitogenome, with r damage . As the r damage . Diverge(mt-cytb . From ou