We conducted experiments in CCM mutant mice by intravenously injecting tozuleristide and analyzin... more We conducted experiments in CCM mutant mice by intravenously injecting tozuleristide and analyzing the binding sites in the mouse brain.
Background: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delaye... more Background: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delayed cognitive deficits. Neutrophil infiltration into the central nervous system (CNS) is linked to the development of these deficits after SAH. It is however unclear how neutrophil activity influences CNS function in SAH. As such, the present project aims to elucidate neutrophil factors and mechanisms mediating CNS injury and cognitive deficits after SAH. Methods: Using a murine model of SAH and mice deficient in neutrophil effector functions, we determined which neutrophil effector function is critical to the development of deficits after SAH. Also, in vitro techniques were used to elucidate how neutrophils affect cellular function of neurons and glia after SAH. Results: Our results show that following SAH, neutrophils infiltrate the meninges, and not the brain parenchyma. Mice lacking functional myeloperoxidase (MPO KO), a neutrophil enzyme, lack both the meningeal neutrophil infiltration and the cognitive deficits associated with SAH. The re-introduction of biologically active MPO, and its substrate hydrogen peroxide, to the cerebrospinal fluid of MPO KO mice at the time of hemorrhage restores the spatial memory deficit observed after SAH. Furthermore, in culture, MPO affects the function of both primary neurons and astrocytes, though not microglia. Neurons exposed to MPO and its substrate show decreased calcium activity at baseline and after stimulation with potassium chloride. In addition, MPO and its substrate lead to significant astrocyte loss in culture, phenocopying a result observed in the brain after SAH. Conclusions: These results implicate MPO as a mediator of neuronal dysfunction in SAH through their effect on both neurons and astrocytes. Finally, these results show that, in SAH, the activity of innate immune cells in the meninges can modulate the activity and function of the underlying brain tissue. increased neutrophil infiltration in the brain in a model of ischemic stroke coincides with increased neuronal loss(3). However, the cellular and molecular mechanism of action of neutrophils in the CNS is poorly understood. Our laboratory has shown that peripheral neutrophil infiltration leads to the development of cognitive deficits in a murine model of subarachnoid hemorrhage (SAH) due to loss of late long-term potentiation (L-LTP) in hippocampal neurons(4,5). Interestingly, although SAH neutrophils do not invade the hippocampus, they never-the-less seem to drive neuronal dysfunction(4).Neutrophils have a limited but potent set of effector functions that are implicated in neurological injuries. These effector functions include enzymes that catalyze the production of reactive oxygen and nitrogen species, and the degradation of extracellular matrix proteins(6,7). A number of these neutrophil enzymes have been linked to brain pathology after injury. For example, myeloperoxidase and elastase (both neutrophil granule enzymes) increase oxidative stress in the brain after cerebral ischemia (8,9). In fact, modulation of myeloperoxidase activity after stroke leads to better behavioral outcomes(9). Limb remote ischemic postconditioning improves stroke outcomes by decreasing the activity of NADPH oxidase and MPO in peripheral neutrophils(10). Inhibition of elastase improves motor recovery and spinal cord damage after injury(11). Although all these results implicate neutrophil proteins in injury-induced neuropathology, what cells the neutrophils interact with to cause neuropathology remains unclear. Study of brain inflammatory pathology in most sterile conditions is limited due to the superimposed necrosis that causes direct brain damage and allows peripheral immune cells to invade the parenchyma thereby mixing the neuroinflammatory response and the necrosis-driven inflammatory invasion. The murine model of subarachnoid hemorrhage is a useful model to study the effect of inflammation on brain physiology because the direct injury occurs outside the brain parenchyma in the meninges. In addition, patients with aneurysmal subarachnoid hemorrhage develop a delayed syndrome of cognitive impairment and vascular spasm(12-15) (called cerebral vasospasm) that the murine model of subarachnoid hemorrhage recapitulates and is a readout of brain injury(16). In this project, we investigate the location and molecular mechanism of action of neutrophils in the central nervous system after SAH. Using a murine model of mild subarachnoid hemorrhage, we 5 demonstrate that neutrophils affect neuronal function without invading the brain parenchyma, providing further evidence of the neuromodulatory role of the peripheral immune system in acute brain injuries. Indeed, our results also demonstrate that modulation of peripheral immune cells affects cells within the brain (i.e. astrocytes and neurons). Finally, we demonstrate that the neutrophil enzyme myeloperoxidase (with its substrate) can affect neuronal function and ultimately modulates neuronal response to stimuli. Materials And Methods Animals Young (8-12 weeks old) male mice were used in all experiments. Mice were kept on a 12 hour:12-hour light cycle at room temperature (22-25 o C). Food and water were provided ad libitum. All experiments were done with the approval of the University of Virginia Animal Care and Use Committee. Transgenic mice deficient in the enzymes elastase (B6.129X1-Elane tm1sds: elastase KO), myeloperoxidase (B6.129X1-Mpo tm1Lus: MPO KO) or neutrophil cytosolic factor 1(NCF-component of the NADPH oxidase complex (B6(Cg)-Bcf1 m1J: NADPH oxidase KO) (The Jackson Laboratories) were used. All transgenic mice used were on a C57BL/6 background; therefore, control experiments were conducted on C57BL/6J mice. The power assessment was completed as follows. In our previous studies, we have shown that significant behavioral changes in mice after SAH compared to control are seen with 6-12 animals(5,16,24). Therefore, a minimum of 6 animals was used for each experimental condition. Animal numbers used in each experiment are included in each of the figures. Subarachnoid hemorrhage (SAH) SAH or sham surgeries were performed as previously reported(16). Briefly, mice were anesthetized using isoflurane, and placed in a prone position. A 3mm incision was made on the back of the neck along the midline, the atlanto-occipital membrane was entered, and a conserved subarachnoid vein punctured. Bleeding from the vein was allowed to stop on its own. For the sham experiment, the procedure was the same except the atlanto-occipital membrane was not entered and the vein was not punctured.
Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by isc... more Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by ischemia and reperfusion. Using murine stroke models of middle cerebral artery occlusion, we demonstrate that it is feasible to deliver viable mitochondria to ischemic brain parenchyma via an intra-arterial route of administration. We also show the beneficial supplemental effects of concurrent focused ultrasound activation of microbubbles, which serves to open the blood-brain barrier without hemorrhagic complications. Following delivery, mitochondria distribute through the stroked hemisphere and integrate into neural and glial cells in the brain parenchyma. Consistent with functional integration in the ischemic tissue, the transplanted mitochondria elevate concentration of adenosine triphosphate in the stroked hemisphere, reduce infarct volume and increase neuronal viability. Our results have implications for the development of interventional strategies after ischemic stroke and suggest a novel potential modality of therapy after mechanical thrombectomy.
Introduction: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothel... more Introduction: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known. Methods: We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice. Results: Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins. Conclusion: Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.
Enhanced recovery after surgery (ERAS) is an evidence-based approach developed to ameliorate the ... more Enhanced recovery after surgery (ERAS) is an evidence-based approach developed to ameliorate the patient recovery process following surgical procedures. Employing a multimodal, multidisciplinary approach, ERAS implements strategies and treatment paradigms that have been shown to improve patient outcomes, reduce hospital length of stay, and ultimately reduce healthcare costs. With a substantial body of the literature supporting the implementation of ERAS in other surgical specialties, ERAS has only recently made its foray into spine surgery. Despite this, current studies are limited to spinal deformity and degenerative disease, with limited data regarding spinal cord surgery. This is due in part to the complex nature and rarity of spinal cord lesions, making the establishment of a formal ERAS protocol difficult. In developing an ERAS protocol, there must be a consensus on what factors are important to consider and implement. To address this, we reviewed the most recent advances in intramedullary and extramedullary spinal cord surgery in order to identify elements that influence patient outcomes. Using this information, the authors provide evidence-based recommendations with the intent of introducing a framework for future ERAS protocols with respect to treating spinal cord lesions.
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, Jul 17, 2017
The rhombomeric compartments of the hindbrain are characterized by lineage restriction; cells bor... more The rhombomeric compartments of the hindbrain are characterized by lineage restriction; cells born in one compartment generally remain there and do not migrate to neighboring rhombomeres. Two well-known exceptions are the substantial migrations of the pontine nuclei and the mammalian facial nucleus. In this study we used Hoxa3-Cre lineage to permanently mark cells that originate in rhombomeres caudal to r4. We found that cells born caudal to the r4/r5 border migrate forwards to a number of different locations in rhombomeres 1-4; the final locations include the interfascicular trigeminal nucleus, the principal trigeminal nucleus, the pontine nuclei, the reticulotegmental nucleus, the ventral nucleus of the lateral lemniscus, and the lateral and medial vestibular nuclei. We suggest that there are numerous exceptions to the principle of rhombomeric lineage restriction that have previously gone unnoticed.
While the majority of cerebral revascularization advancements were made in the last century, it i... more While the majority of cerebral revascularization advancements were made in the last century, it is worth noting the humble beginnings of vascular surgery throughout history to appreciate its progression and application to neurovascular pathology in the modern era. Nearly 5000 years of basic human inquiry into the vasculature and its role in neurological disease has resulted in the complex neurosurgical procedures used today to save and improve lives. This paper explores the story of the extracranial-intracranial approach to cerebral revascularization.
Establishing blood vessel patency in neurovascular surgery is an essential component in treating ... more Establishing blood vessel patency in neurovascular surgery is an essential component in treating cerebrovascular disorders. Given the difficulty in confirming complete obliteration of the aneurysm sac, ICG videoangiography has emerged as an intraoperative tool that provides neurosurgeons immediate feedback on the status of vessel flow, allowing for surgical modifications to be made without delay. ICG initially emerged as a tool for assessing hepatic, cardiac, and retinovascular function. It is an inert compound with a high affinity for plasma proteins and fluorescence properties making it the ideal candidate for assessment of vessel patency in neurovascular procedures. Requiring only a bolus peripheral vein injection and integration of a near-infrared imaging device into the surgical microscope, ICG can be visualized without disrupting operating room workflow or the surgical field. Quick response time, high-spatial resolution, and low complication rates are features of ICG videoangiography that prove advantageous when compared to the gold standard intra-and postoperative digital subtraction angiography (DSA). Despite this, ICG is not without limitations, specifically in the setting of atherosclerotic vessels, giant, and complex aneurysms. Additionally, there are instances where DSA may prove superior in detecting vessel stenosis and outflow obstruction, prompting the recommendation of ICG as an adjunct to, rather than complete replacement of DSA. In this article, the authors provide a brief overview of the biochemical properties and historical origins of ICG viedoangiography in addition to discussing its current application in aneurysm surgery.
In order to examine how norepinephrine stimulates proliferation and differentiation in brown fat ... more In order to examine how norepinephrine stimulates proliferation and differentiation in brown fat cells, we have investigated the ability of brown fat cells to respond to norepinephrine stimulation with an increase in the expression of the proto-oncogene c-fos. Stimulation of brown fat precursor cells (isolated from young mice and grown for 4 days in culture) with norepinephrine led to a marked but transient (maximal approximately 30 min) induction of c-fos expression. The magnitude of this induction was similar in pre- and postconfluent cells. The norepinephrine effect could be blocked by both alpha 1- and beta-adrenergic antagonists. Forskolin had a small inductive ability, as had the selective alpha 1-agonist cirazoline, but with both together a high induction was obtained. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) could in itself induce c-fos expression, but pretreatment with TPA did not abolish the ability of norepinephrine to induce c-fos expression, indicating that TPA-sensitive protein kinase C was not a primary mediator in this pathway. Also the Ca2+ ionophore A23187 had in itself an inductive ability, but A23187 in combination with forskolin led to a large increase in c-fos expression, indicating synergistic interaction between a cAMP pathway and a [Ca2+]i pathway. This interaction was not proximal, i.e. alpha 1 stimulation or increase in [Ca2+]i by A23187 did not augment forskolin-induced cAMP levels, and beta stimulation or forskolin did not affect [Ca2+]i levels; and it did not require protein synthesis. It was concluded that norepinephrine, in agreement with its fundamental role in the control of brown fat cell growth and development, was able to induce c-fos expression, that this induction was not exclusively linked to promotion of either proliferation or differentiation, and that the induction was mediated via a distal synergism between beta/cAMP and alpha 1/[Ca2+]i pathways, thus conferring to the alpha 1-adrenoreceptors on the cell a potentially significant role in the control of cell growth and development.
Mitochondria are fundamental for metabolic homeostasis in all multicellular eukaryotes. In the ne... more Mitochondria are fundamental for metabolic homeostasis in all multicellular eukaryotes. In the nervous system, mitochondriagenerated adenosine triphosphate (ATP) is required to establish appropriate electrochemical gradients and reliable synaptic transmission. Notably, several mitochondrial defects have been identified in central nervous system disorders. Membrane leakage and electrolyte imbalances, pro-apoptotic pathway activation, and mitophagy are among the mechanisms implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's disease, as well as ischemic stroke. In this review, we summarize mitochondrial pathways that contribute to disease progression. Further, we discuss pathological states that damaged mitochondria impose on normal nervous system processes and explore new therapeutic approaches to mitochondrial diseases.
Arteriovenous malformation (AVM) presenting with epilepsy significantly impacts patient quality o... more Arteriovenous malformation (AVM) presenting with epilepsy significantly impacts patient quality of life, and it should be considered very much a seizure disorder. Although hemorrhage prevention is the primary treatment aim of AVM surgery, seizure control should also be at the forefront of therapeutic management. Several hemodynamic and morphological characteristics of AVM have been identified to be associated with seizure presentation. This includes increased AVM flow, presence of long pial draining vein, venous outflow obstruction, and frontotemporal location, among other aspects. With the advent of high-throughput image processing and quantification methods, new radiographic attributes of AVMrelated epilepsy have been identified. With respect to therapy, several treatment approaches are available, including conservative management or interventional modalities; this includes microsurgery, radiosurgery, and embolization or a combination thereof. Many studies, especially in the domain of microsurgery and radiosurgery, evaluate both techniques with respect to seizure outcomes. The advantage of microsurgery lies in superior AVM obliteration rates and swift seizure response. In addition, by incorporating electrophysiological monitoring during AVM resection, adjacent or even remote epileptogenic foci can be identified, leading to extended lesionectomy and improved seizure control. Radiosurgery, despite resulting in reduced AVM obliteration and prolonged time to seizure freedom, avoids the risks of surgery altogether and may provide seizure control through various antiepileptic mechanisms. Embolization continues to be used as an adjuvant for both microsurgery and radiosurgery. In this study, the authors review the latest imaging techniques in characterizing AVM-related epilepsy, in addition to reviewing each treatment modality.
B rain arteriovenous malformations (AVMs) are characterized by abnormal, high-flow connections be... more B rain arteriovenous malformations (AVMs) are characterized by abnormal, high-flow connections between feeding arteries and draining veins with an intervening plexus of abnormal vessels, which comprises the nidus. 17,41 Direct intranidal arteriovenous fistulas can also be found within AVMs, and the relative contribution of fistulous versus nidal components varies among AVMs. 4,24 The most common presentations of AVMs include hemorrhages, seizures, headaches, and progressive neurological deficits due to chronic vascular steal. 1,13,14 The majority of AVM-associated morbidity and mortality can be attributed to hemorrhage secondary to rupture of the nidus or flow-related aneurysms. 3,16,47 The inherent lack of normal smooth muscle properties, as a result of aberrant angiogenesis and vascular remodeling in the vascular beds of AVMs, predisposes these lesions to rupture. 4,30,55 Risk factors for AVM rupture include prior hemorrhage, associated arterial aneurysms, exclusively deep venous drainage, and deep AVM location. 19,28 Despite this, elimination of hemorrhage risk by extirpation or endoluminal closure of the nidus remains the primary goal of AVM treatment. 11,32,52,53 Contemporary management strategies are dictated by the angioarchitecture, location, and presentation of AVMs. 5,27 Currently employed treatment options include microsurgery, stereotactic radiosurgery, and endovascular embolization as standalone or combination therapies. 12,15,18,31,45 Despite unresolved controversies regarding the choice of treatment modality, obliteration of the AVM nidus with preservation of normal venous drainage remains ABBREVIATIONS AVM = arteriovenous malformation; DAVF = dural arteriovenous fistula; DSA = digital subtraction angiography; ICV = internal cerebral vein; mRS = modified Rankin Scale; NBCA = N-butyl cyanoacrylate; PHIL = precipitating hydrophobic injectable liquid; TRENSH = transvenous retrograde nidus sclerotherapy under controlled hypotension.
Glioma continues to be a challenging disease process, making up the most common tumor type within... more Glioma continues to be a challenging disease process, making up the most common tumor type within the pediatric population. While low-grade gliomas are typically amenable to surgical resection, higher grade gliomas often require additional radiotherapy in conjunction with adjuvant chemotherapy. Molecular profiling of these lesions has led to the development of various pharmacologic and immunologic agents, although these modalities are not without great systemic toxicity. In addition, the molecular biology of adult glioma and pediatric glioma has been shown to differ substantially, making the application of current chemotherapies dubious in children and adolescents. For this reason, therapies with high tumor specificity based on pediatric tumor cell biology that spare healthy tissue are needed. Oncolytic virotherapy serves to fill this niche, as evidenced by renewed interest in this domain of cancer therapy. Initially discovered by chance in the early 20th century, virotherapy has emerged as a viable treatment option. With promising results based on preclinical studies, the authors review several oncolytic viruses, with a focus on molecular mechanism and efficacy of these viruses in tumor cell lines and murine models. In addition, current phase I clinical trials evaluating oncolytic virotherapy in the treatment of pediatric glioma are summarized.
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, May 2, 2018
The classic columnar model of cranial nerve central representation assumes that all motor and sen... more The classic columnar model of cranial nerve central representation assumes that all motor and sensory hindbrain neurons develop within four radial migration domains, held to be separated by a sulcal alar-basal boundary (sulcus limitans). This essay reviews a number of developmental data that challenge these concepts. These results are interpreted within the framework of present day neuromeric conception of the brainstem (the prosomeric model). Advances in dorsoventral patterning of the spinal cord and hindbrain now show that there exist up to eight alar microzones and five basal microzones (molecularly and histogenetically distinct longitudinal progenitor domains). This reveals that the classic tetracolumnar model is excessively simplistic. There is both older and recent data revealing that the visceral efferent neurons of the cranial nerves (preganglionic and branchiomotor neurons) are generated next to the floor plate and later migrate dorsalwards before adopting their final topography in the mantle, contrary to the purely radial migration assumed in the classic model. Moreover, various results support the conclusion that at least the branchiomotor neurons end their migration and mature within the alar region of the mantle. Evidence on this point obtained in chick embryos is reviewed in detail, and novel evidence in mouse embryos is presented.
I ntracranIal aneurysms (IAs) are acquired outpouchings of arteries that occur in 1%-2% of the po... more I ntracranIal aneurysms (IAs) are acquired outpouchings of arteries that occur in 1%-2% of the population. 36 Likely as a result of improved imaging modalities, the incidence of unruptured IAs has increased. Despite the estimated 1% annual risk of rupture, our ability to predict which aneurysms are best managed conservatively versus treated with either open or endovascular techniques is not without controversy. 4,65 While aneurysm size and location are the main determinants in our decision-making process, there are a multitude of additional factors that are often considered. 17,63 Recently, considerable research has focused on the relationship between hemodynamic stressors and aneurysm pathogenesis. From a mathematical and biophysical standpoint, aneurysm morphology and evolution over time is highly complex due to the heterogeneous nature of fluid mechanics. Complicating matters even further is the living nature of blood vessels, i.e., mechanical stimuli are transduced into biological signals, triggering inflammatory cascades and, ultimately, a wide range of transcriptional and signaling changes that lead to vascular wall remodeling. The advent of computational and radiographic modeling has allowed for the study of hemodynamics within vessels and aneurysms. For this reason, cerebral aneurysm hemodynamics can serve as a tool in understanding the biophysical pathogenesis of IAs and has been used to evaluate aneurysm rupture risk. In this article, we review the hemodynamic parameters related to aneurysm formation and rupture and the biophysical pathways that contribute to vascular remodeling. Additionally, we discuss the current state and utility of radiographic and computational modeling in predicting IA rupture in clinical practice.
We conducted experiments in CCM mutant mice by intravenously injecting tozuleristide and analyzin... more We conducted experiments in CCM mutant mice by intravenously injecting tozuleristide and analyzing the binding sites in the mouse brain.
Background: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delaye... more Background: Aneurysmal subarachnoid hemorrhage (SAH) is associated with the development of delayed cognitive deficits. Neutrophil infiltration into the central nervous system (CNS) is linked to the development of these deficits after SAH. It is however unclear how neutrophil activity influences CNS function in SAH. As such, the present project aims to elucidate neutrophil factors and mechanisms mediating CNS injury and cognitive deficits after SAH. Methods: Using a murine model of SAH and mice deficient in neutrophil effector functions, we determined which neutrophil effector function is critical to the development of deficits after SAH. Also, in vitro techniques were used to elucidate how neutrophils affect cellular function of neurons and glia after SAH. Results: Our results show that following SAH, neutrophils infiltrate the meninges, and not the brain parenchyma. Mice lacking functional myeloperoxidase (MPO KO), a neutrophil enzyme, lack both the meningeal neutrophil infiltration and the cognitive deficits associated with SAH. The re-introduction of biologically active MPO, and its substrate hydrogen peroxide, to the cerebrospinal fluid of MPO KO mice at the time of hemorrhage restores the spatial memory deficit observed after SAH. Furthermore, in culture, MPO affects the function of both primary neurons and astrocytes, though not microglia. Neurons exposed to MPO and its substrate show decreased calcium activity at baseline and after stimulation with potassium chloride. In addition, MPO and its substrate lead to significant astrocyte loss in culture, phenocopying a result observed in the brain after SAH. Conclusions: These results implicate MPO as a mediator of neuronal dysfunction in SAH through their effect on both neurons and astrocytes. Finally, these results show that, in SAH, the activity of innate immune cells in the meninges can modulate the activity and function of the underlying brain tissue. increased neutrophil infiltration in the brain in a model of ischemic stroke coincides with increased neuronal loss(3). However, the cellular and molecular mechanism of action of neutrophils in the CNS is poorly understood. Our laboratory has shown that peripheral neutrophil infiltration leads to the development of cognitive deficits in a murine model of subarachnoid hemorrhage (SAH) due to loss of late long-term potentiation (L-LTP) in hippocampal neurons(4,5). Interestingly, although SAH neutrophils do not invade the hippocampus, they never-the-less seem to drive neuronal dysfunction(4).Neutrophils have a limited but potent set of effector functions that are implicated in neurological injuries. These effector functions include enzymes that catalyze the production of reactive oxygen and nitrogen species, and the degradation of extracellular matrix proteins(6,7). A number of these neutrophil enzymes have been linked to brain pathology after injury. For example, myeloperoxidase and elastase (both neutrophil granule enzymes) increase oxidative stress in the brain after cerebral ischemia (8,9). In fact, modulation of myeloperoxidase activity after stroke leads to better behavioral outcomes(9). Limb remote ischemic postconditioning improves stroke outcomes by decreasing the activity of NADPH oxidase and MPO in peripheral neutrophils(10). Inhibition of elastase improves motor recovery and spinal cord damage after injury(11). Although all these results implicate neutrophil proteins in injury-induced neuropathology, what cells the neutrophils interact with to cause neuropathology remains unclear. Study of brain inflammatory pathology in most sterile conditions is limited due to the superimposed necrosis that causes direct brain damage and allows peripheral immune cells to invade the parenchyma thereby mixing the neuroinflammatory response and the necrosis-driven inflammatory invasion. The murine model of subarachnoid hemorrhage is a useful model to study the effect of inflammation on brain physiology because the direct injury occurs outside the brain parenchyma in the meninges. In addition, patients with aneurysmal subarachnoid hemorrhage develop a delayed syndrome of cognitive impairment and vascular spasm(12-15) (called cerebral vasospasm) that the murine model of subarachnoid hemorrhage recapitulates and is a readout of brain injury(16). In this project, we investigate the location and molecular mechanism of action of neutrophils in the central nervous system after SAH. Using a murine model of mild subarachnoid hemorrhage, we 5 demonstrate that neutrophils affect neuronal function without invading the brain parenchyma, providing further evidence of the neuromodulatory role of the peripheral immune system in acute brain injuries. Indeed, our results also demonstrate that modulation of peripheral immune cells affects cells within the brain (i.e. astrocytes and neurons). Finally, we demonstrate that the neutrophil enzyme myeloperoxidase (with its substrate) can affect neuronal function and ultimately modulates neuronal response to stimuli. Materials And Methods Animals Young (8-12 weeks old) male mice were used in all experiments. Mice were kept on a 12 hour:12-hour light cycle at room temperature (22-25 o C). Food and water were provided ad libitum. All experiments were done with the approval of the University of Virginia Animal Care and Use Committee. Transgenic mice deficient in the enzymes elastase (B6.129X1-Elane tm1sds: elastase KO), myeloperoxidase (B6.129X1-Mpo tm1Lus: MPO KO) or neutrophil cytosolic factor 1(NCF-component of the NADPH oxidase complex (B6(Cg)-Bcf1 m1J: NADPH oxidase KO) (The Jackson Laboratories) were used. All transgenic mice used were on a C57BL/6 background; therefore, control experiments were conducted on C57BL/6J mice. The power assessment was completed as follows. In our previous studies, we have shown that significant behavioral changes in mice after SAH compared to control are seen with 6-12 animals(5,16,24). Therefore, a minimum of 6 animals was used for each experimental condition. Animal numbers used in each experiment are included in each of the figures. Subarachnoid hemorrhage (SAH) SAH or sham surgeries were performed as previously reported(16). Briefly, mice were anesthetized using isoflurane, and placed in a prone position. A 3mm incision was made on the back of the neck along the midline, the atlanto-occipital membrane was entered, and a conserved subarachnoid vein punctured. Bleeding from the vein was allowed to stop on its own. For the sham experiment, the procedure was the same except the atlanto-occipital membrane was not entered and the vein was not punctured.
Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by isc... more Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by ischemia and reperfusion. Using murine stroke models of middle cerebral artery occlusion, we demonstrate that it is feasible to deliver viable mitochondria to ischemic brain parenchyma via an intra-arterial route of administration. We also show the beneficial supplemental effects of concurrent focused ultrasound activation of microbubbles, which serves to open the blood-brain barrier without hemorrhagic complications. Following delivery, mitochondria distribute through the stroked hemisphere and integrate into neural and glial cells in the brain parenchyma. Consistent with functional integration in the ischemic tissue, the transplanted mitochondria elevate concentration of adenosine triphosphate in the stroked hemisphere, reduce infarct volume and increase neuronal viability. Our results have implications for the development of interventional strategies after ischemic stroke and suggest a novel potential modality of therapy after mechanical thrombectomy.
Introduction: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothel... more Introduction: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known. Methods: We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice. Results: Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins. Conclusion: Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.
Enhanced recovery after surgery (ERAS) is an evidence-based approach developed to ameliorate the ... more Enhanced recovery after surgery (ERAS) is an evidence-based approach developed to ameliorate the patient recovery process following surgical procedures. Employing a multimodal, multidisciplinary approach, ERAS implements strategies and treatment paradigms that have been shown to improve patient outcomes, reduce hospital length of stay, and ultimately reduce healthcare costs. With a substantial body of the literature supporting the implementation of ERAS in other surgical specialties, ERAS has only recently made its foray into spine surgery. Despite this, current studies are limited to spinal deformity and degenerative disease, with limited data regarding spinal cord surgery. This is due in part to the complex nature and rarity of spinal cord lesions, making the establishment of a formal ERAS protocol difficult. In developing an ERAS protocol, there must be a consensus on what factors are important to consider and implement. To address this, we reviewed the most recent advances in intramedullary and extramedullary spinal cord surgery in order to identify elements that influence patient outcomes. Using this information, the authors provide evidence-based recommendations with the intent of introducing a framework for future ERAS protocols with respect to treating spinal cord lesions.
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, Jul 17, 2017
The rhombomeric compartments of the hindbrain are characterized by lineage restriction; cells bor... more The rhombomeric compartments of the hindbrain are characterized by lineage restriction; cells born in one compartment generally remain there and do not migrate to neighboring rhombomeres. Two well-known exceptions are the substantial migrations of the pontine nuclei and the mammalian facial nucleus. In this study we used Hoxa3-Cre lineage to permanently mark cells that originate in rhombomeres caudal to r4. We found that cells born caudal to the r4/r5 border migrate forwards to a number of different locations in rhombomeres 1-4; the final locations include the interfascicular trigeminal nucleus, the principal trigeminal nucleus, the pontine nuclei, the reticulotegmental nucleus, the ventral nucleus of the lateral lemniscus, and the lateral and medial vestibular nuclei. We suggest that there are numerous exceptions to the principle of rhombomeric lineage restriction that have previously gone unnoticed.
While the majority of cerebral revascularization advancements were made in the last century, it i... more While the majority of cerebral revascularization advancements were made in the last century, it is worth noting the humble beginnings of vascular surgery throughout history to appreciate its progression and application to neurovascular pathology in the modern era. Nearly 5000 years of basic human inquiry into the vasculature and its role in neurological disease has resulted in the complex neurosurgical procedures used today to save and improve lives. This paper explores the story of the extracranial-intracranial approach to cerebral revascularization.
Establishing blood vessel patency in neurovascular surgery is an essential component in treating ... more Establishing blood vessel patency in neurovascular surgery is an essential component in treating cerebrovascular disorders. Given the difficulty in confirming complete obliteration of the aneurysm sac, ICG videoangiography has emerged as an intraoperative tool that provides neurosurgeons immediate feedback on the status of vessel flow, allowing for surgical modifications to be made without delay. ICG initially emerged as a tool for assessing hepatic, cardiac, and retinovascular function. It is an inert compound with a high affinity for plasma proteins and fluorescence properties making it the ideal candidate for assessment of vessel patency in neurovascular procedures. Requiring only a bolus peripheral vein injection and integration of a near-infrared imaging device into the surgical microscope, ICG can be visualized without disrupting operating room workflow or the surgical field. Quick response time, high-spatial resolution, and low complication rates are features of ICG videoangiography that prove advantageous when compared to the gold standard intra-and postoperative digital subtraction angiography (DSA). Despite this, ICG is not without limitations, specifically in the setting of atherosclerotic vessels, giant, and complex aneurysms. Additionally, there are instances where DSA may prove superior in detecting vessel stenosis and outflow obstruction, prompting the recommendation of ICG as an adjunct to, rather than complete replacement of DSA. In this article, the authors provide a brief overview of the biochemical properties and historical origins of ICG viedoangiography in addition to discussing its current application in aneurysm surgery.
In order to examine how norepinephrine stimulates proliferation and differentiation in brown fat ... more In order to examine how norepinephrine stimulates proliferation and differentiation in brown fat cells, we have investigated the ability of brown fat cells to respond to norepinephrine stimulation with an increase in the expression of the proto-oncogene c-fos. Stimulation of brown fat precursor cells (isolated from young mice and grown for 4 days in culture) with norepinephrine led to a marked but transient (maximal approximately 30 min) induction of c-fos expression. The magnitude of this induction was similar in pre- and postconfluent cells. The norepinephrine effect could be blocked by both alpha 1- and beta-adrenergic antagonists. Forskolin had a small inductive ability, as had the selective alpha 1-agonist cirazoline, but with both together a high induction was obtained. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) could in itself induce c-fos expression, but pretreatment with TPA did not abolish the ability of norepinephrine to induce c-fos expression, indicating that TPA-sensitive protein kinase C was not a primary mediator in this pathway. Also the Ca2+ ionophore A23187 had in itself an inductive ability, but A23187 in combination with forskolin led to a large increase in c-fos expression, indicating synergistic interaction between a cAMP pathway and a [Ca2+]i pathway. This interaction was not proximal, i.e. alpha 1 stimulation or increase in [Ca2+]i by A23187 did not augment forskolin-induced cAMP levels, and beta stimulation or forskolin did not affect [Ca2+]i levels; and it did not require protein synthesis. It was concluded that norepinephrine, in agreement with its fundamental role in the control of brown fat cell growth and development, was able to induce c-fos expression, that this induction was not exclusively linked to promotion of either proliferation or differentiation, and that the induction was mediated via a distal synergism between beta/cAMP and alpha 1/[Ca2+]i pathways, thus conferring to the alpha 1-adrenoreceptors on the cell a potentially significant role in the control of cell growth and development.
Mitochondria are fundamental for metabolic homeostasis in all multicellular eukaryotes. In the ne... more Mitochondria are fundamental for metabolic homeostasis in all multicellular eukaryotes. In the nervous system, mitochondriagenerated adenosine triphosphate (ATP) is required to establish appropriate electrochemical gradients and reliable synaptic transmission. Notably, several mitochondrial defects have been identified in central nervous system disorders. Membrane leakage and electrolyte imbalances, pro-apoptotic pathway activation, and mitophagy are among the mechanisms implicated in the pathogenesis of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's disease, as well as ischemic stroke. In this review, we summarize mitochondrial pathways that contribute to disease progression. Further, we discuss pathological states that damaged mitochondria impose on normal nervous system processes and explore new therapeutic approaches to mitochondrial diseases.
Arteriovenous malformation (AVM) presenting with epilepsy significantly impacts patient quality o... more Arteriovenous malformation (AVM) presenting with epilepsy significantly impacts patient quality of life, and it should be considered very much a seizure disorder. Although hemorrhage prevention is the primary treatment aim of AVM surgery, seizure control should also be at the forefront of therapeutic management. Several hemodynamic and morphological characteristics of AVM have been identified to be associated with seizure presentation. This includes increased AVM flow, presence of long pial draining vein, venous outflow obstruction, and frontotemporal location, among other aspects. With the advent of high-throughput image processing and quantification methods, new radiographic attributes of AVMrelated epilepsy have been identified. With respect to therapy, several treatment approaches are available, including conservative management or interventional modalities; this includes microsurgery, radiosurgery, and embolization or a combination thereof. Many studies, especially in the domain of microsurgery and radiosurgery, evaluate both techniques with respect to seizure outcomes. The advantage of microsurgery lies in superior AVM obliteration rates and swift seizure response. In addition, by incorporating electrophysiological monitoring during AVM resection, adjacent or even remote epileptogenic foci can be identified, leading to extended lesionectomy and improved seizure control. Radiosurgery, despite resulting in reduced AVM obliteration and prolonged time to seizure freedom, avoids the risks of surgery altogether and may provide seizure control through various antiepileptic mechanisms. Embolization continues to be used as an adjuvant for both microsurgery and radiosurgery. In this study, the authors review the latest imaging techniques in characterizing AVM-related epilepsy, in addition to reviewing each treatment modality.
B rain arteriovenous malformations (AVMs) are characterized by abnormal, high-flow connections be... more B rain arteriovenous malformations (AVMs) are characterized by abnormal, high-flow connections between feeding arteries and draining veins with an intervening plexus of abnormal vessels, which comprises the nidus. 17,41 Direct intranidal arteriovenous fistulas can also be found within AVMs, and the relative contribution of fistulous versus nidal components varies among AVMs. 4,24 The most common presentations of AVMs include hemorrhages, seizures, headaches, and progressive neurological deficits due to chronic vascular steal. 1,13,14 The majority of AVM-associated morbidity and mortality can be attributed to hemorrhage secondary to rupture of the nidus or flow-related aneurysms. 3,16,47 The inherent lack of normal smooth muscle properties, as a result of aberrant angiogenesis and vascular remodeling in the vascular beds of AVMs, predisposes these lesions to rupture. 4,30,55 Risk factors for AVM rupture include prior hemorrhage, associated arterial aneurysms, exclusively deep venous drainage, and deep AVM location. 19,28 Despite this, elimination of hemorrhage risk by extirpation or endoluminal closure of the nidus remains the primary goal of AVM treatment. 11,32,52,53 Contemporary management strategies are dictated by the angioarchitecture, location, and presentation of AVMs. 5,27 Currently employed treatment options include microsurgery, stereotactic radiosurgery, and endovascular embolization as standalone or combination therapies. 12,15,18,31,45 Despite unresolved controversies regarding the choice of treatment modality, obliteration of the AVM nidus with preservation of normal venous drainage remains ABBREVIATIONS AVM = arteriovenous malformation; DAVF = dural arteriovenous fistula; DSA = digital subtraction angiography; ICV = internal cerebral vein; mRS = modified Rankin Scale; NBCA = N-butyl cyanoacrylate; PHIL = precipitating hydrophobic injectable liquid; TRENSH = transvenous retrograde nidus sclerotherapy under controlled hypotension.
Glioma continues to be a challenging disease process, making up the most common tumor type within... more Glioma continues to be a challenging disease process, making up the most common tumor type within the pediatric population. While low-grade gliomas are typically amenable to surgical resection, higher grade gliomas often require additional radiotherapy in conjunction with adjuvant chemotherapy. Molecular profiling of these lesions has led to the development of various pharmacologic and immunologic agents, although these modalities are not without great systemic toxicity. In addition, the molecular biology of adult glioma and pediatric glioma has been shown to differ substantially, making the application of current chemotherapies dubious in children and adolescents. For this reason, therapies with high tumor specificity based on pediatric tumor cell biology that spare healthy tissue are needed. Oncolytic virotherapy serves to fill this niche, as evidenced by renewed interest in this domain of cancer therapy. Initially discovered by chance in the early 20th century, virotherapy has emerged as a viable treatment option. With promising results based on preclinical studies, the authors review several oncolytic viruses, with a focus on molecular mechanism and efficacy of these viruses in tumor cell lines and murine models. In addition, current phase I clinical trials evaluating oncolytic virotherapy in the treatment of pediatric glioma are summarized.
Anatomical Record-advances in Integrative Anatomy and Evolutionary Biology, May 2, 2018
The classic columnar model of cranial nerve central representation assumes that all motor and sen... more The classic columnar model of cranial nerve central representation assumes that all motor and sensory hindbrain neurons develop within four radial migration domains, held to be separated by a sulcal alar-basal boundary (sulcus limitans). This essay reviews a number of developmental data that challenge these concepts. These results are interpreted within the framework of present day neuromeric conception of the brainstem (the prosomeric model). Advances in dorsoventral patterning of the spinal cord and hindbrain now show that there exist up to eight alar microzones and five basal microzones (molecularly and histogenetically distinct longitudinal progenitor domains). This reveals that the classic tetracolumnar model is excessively simplistic. There is both older and recent data revealing that the visceral efferent neurons of the cranial nerves (preganglionic and branchiomotor neurons) are generated next to the floor plate and later migrate dorsalwards before adopting their final topography in the mantle, contrary to the purely radial migration assumed in the classic model. Moreover, various results support the conclusion that at least the branchiomotor neurons end their migration and mature within the alar region of the mantle. Evidence on this point obtained in chick embryos is reviewed in detail, and novel evidence in mouse embryos is presented.
I ntracranIal aneurysms (IAs) are acquired outpouchings of arteries that occur in 1%-2% of the po... more I ntracranIal aneurysms (IAs) are acquired outpouchings of arteries that occur in 1%-2% of the population. 36 Likely as a result of improved imaging modalities, the incidence of unruptured IAs has increased. Despite the estimated 1% annual risk of rupture, our ability to predict which aneurysms are best managed conservatively versus treated with either open or endovascular techniques is not without controversy. 4,65 While aneurysm size and location are the main determinants in our decision-making process, there are a multitude of additional factors that are often considered. 17,63 Recently, considerable research has focused on the relationship between hemodynamic stressors and aneurysm pathogenesis. From a mathematical and biophysical standpoint, aneurysm morphology and evolution over time is highly complex due to the heterogeneous nature of fluid mechanics. Complicating matters even further is the living nature of blood vessels, i.e., mechanical stimuli are transduced into biological signals, triggering inflammatory cascades and, ultimately, a wide range of transcriptional and signaling changes that lead to vascular wall remodeling. The advent of computational and radiographic modeling has allowed for the study of hemodynamics within vessels and aneurysms. For this reason, cerebral aneurysm hemodynamics can serve as a tool in understanding the biophysical pathogenesis of IAs and has been used to evaluate aneurysm rupture risk. In this article, we review the hemodynamic parameters related to aneurysm formation and rupture and the biophysical pathways that contribute to vascular remodeling. Additionally, we discuss the current state and utility of radiographic and computational modeling in predicting IA rupture in clinical practice.
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Papers by Petr Tvrdik