Ventral Pons Research Articles

18 kDa Translocator protein (TSPO) is upregulated in rat brain after peripheral nerve injury and downregulated by diroximel fumarate

Neuroimmune signaling is a key process underlying neuropathic pain. Clinical studies have demonstrated that 18 kDa translocator protein (TSPO), a putative marker of neuroinflammation, is upregulated in discrete brain regions of patients with chronic pain. However, no preclinical studies have investigated TSPO dynamics in the brain in the context of neuropathic pain and in response to analgesic treatments. We used positron emission tomography-computed tomography (PET-CT) and [18F]-PBR06 radioligand to measure TSPO levels in the brain across time after chronic constriction injury (CCI) of the sciatic nerve in both male and female rats. Up to 10 weeks post-CCI, TSPO expression was increased in discrete brain regions, including medial prefrontal cortex, somatosensory cortex, insular cortex, anterior cingulate cortex, motor cortex, ventral tegmental area, amygdala, midbrain, pons, medulla, and nucleus accumbens. TSPO was broadly upregulated across these regions at 4 weeks post CCI in males, and 10 weeks in females, though there were regional differences between the sexes. Using immunohistochemistry, we confirmed TSPO expression in these regions. We further demonstrated that TSPO was upregulated principally in microglia in the nucleus accumbens core, and astrocytes and endothelial cells in the nucleus accumbens shell. Finally, we tested whether TSPO upregulation was sensitive to diroximel fumarate, a drug that induces endogenous antioxidants via nuclear factor E2-related factor 2 (Nrf2). Diroximel fumarate alleviated neuropathic pain and reduced TSPO upregulation. Our findings indicate that TSPO is upregulated over the course of neuropathic pain development and is resolved by an antinociceptive intervention in animals with peripheral nerve injury.

Anomalies of Midbrain Hindbrain Development: Midbrain Clefts, Cerebellar Nodular Heterotopia with Overlying Dysgenesis, Cerebellar Foliation Disorder, Pontine Tegmental Cap Dysplasia; Joubert Syndrome; Lhermitte Duclos Syndrome. Diagnosis, Classification and Rehabilitation Hypothesis

AbstractMidbrain and hindbrain (MBHB) malformations are a rare group of congenital abnormalities that involve the neural structure of the posterior cranial fossa, leading to significant causes of neurodevelopmental dysfunction. Recent advancements in genetic and neuroimaging technologies have significantly enhanced our understanding of these disorders. The integration of these advances has facilitated a systematic classification of these conditions. A basic understanding of MBHB embryology is fundamental in order to understand the malformations occurring in their structures: MBHB neurons are mainly generated in the neuroepithelium, lining the walls of the fourth ventricle. Moreover, the regional specificity of the neural tube is determined by a combination of transcription factors expressed, organizing the fate of the neighboring regions as well. Clinical features of MBHB malformations are typically nonspecific; some patients may be asymptomatic or may develop neurological symptoms including hypotonia, ataxia, abnormal eye movements, decreased visual attention, cranial nerve deficits, cognitive impairment, and psychiatric symptoms. Many malformations have been described. We proposed the description of some of them, reporting their main morphologic aspects, magnetic resonance imaging (MRI) peculiar signs and their clinical presentation. Midbrain clefts, for example, are malformations characterized by median separation in the ventral midbrain which involves a communication with the cerebral aqueduct giving a “keyhole” shape. Pontine tegmental cap dysplasia, instead, is a rare hindbrain malformation responsible for a nonprogressive neurological disorder and is described with hypoplastic flat ventral pons, hypoplasia of the middle cerebellar peduncles, and hypoplasia and malformation of the worm. Joubert syndrome, cerebellar nodular heterotopia, abnormal cerebellar foliation, and Lhermitte–Duclos disease, also called dysplastic cerebellar gangliocytoma, have been described as well in order to provide a general overview on this diagnostic challenge reporting the most recent findings.

Anomalies of Midbrain/Hindbrain Development and Related Disabilities: Pontocerebellar Hypoplasia, Congenital Disorders of Glycosylation, and Cerebellar Hemisphere Hypoplasia

AbstractRecent progress in developmental biology, molecular genetics, and neuroimaging has enabled a more profound comprehension of developmental disorders affecting the embryonic midbrain and hindbrain, which manifest clinically. The purpose of this review is to describe anomalies of the midbrain/hindbrain such as pontocerebellar hypoplasia (PCH), congenital disorders of glycosylation (CDG), cerebellar hemisphere hypoplasia. PCH is a group of disorders that is both clinically and genetically diverse. These disorders are identified by the hypoplasia and degeneration of the cerebellum and ventral pons. A total of 18 distinct clinical subtypes of PCH, each linked to pathogenic variants in 19 different genes, have been documented, like mutations in TSEN54 (coding a subunit of tRNA splicing endonucleases complex) and TBC1D23 which display moderate-to-severe intellectual disability (ID) and microcephaly. CDG represent a set of inherited conditions marked by impaired glycosylation of proteins and lipids. The most prevalent subtype among CDG is PMM2-CDG, inherited in a recessive manner, causing reduced activity of phosphomannomutase. Its phenotype varies from mild to severe, involving the central nervous system and affecting many other organs as well. Patients who are severely affected also exhibit visceral symptoms alongside severe ID and other neurological manifestations. Cerebellar hypoplasia (CH) is characterized by a cerebellum of diminished volume while maintaining its shape. CH exhibits a diverse range of neuroradiologic features, etiologies, clinical characteristics, and neurodevelopmental involvement. Cerebello–oculo–facio–genital syndrome is linked to a recessive MAB21L1 mutation. Jubert's syndrome, associated with a rare autosomal recessive mutation, is identified on magnetic resonance imaging by cerebellar worm hypoplasia and midbrain malformations. The rhombencephalosynapsis, characterized by vermian agenesis or hypogenesis with the fusion of the cerebellar hemispheres, emerges during embryogenesis. It can manifest alone or in conjunction with other and/or extracerebral abnormalities.

438 Lesion Combination on Susceptibility-Weighted MRI Predicts Functional Recovery After Diffuse Axonal Injury

INTRODUCTION: Early prediction of long-term functional outcome after diffuse axonal injury (DAI) remains controversial. METHODS: In a retrospective observational study, axonal injuries on MRI were correlated with long-term functional outcome. The predictive value of poor functional outcome (GOSE < 5) was determined by ROC analysis for clinical factors and location of combinations of DAI lesions in the connectome. RESULTS: Over a 10-year period, 451 patients underwent MRI within 30 days of severe blunt TBI of which 322 (71.4%) survived to = 6-month clinical follow-up. The mean age was 38.4 and 242 (75%) patients were male. Mechanisms of injury were motor vehicle crash (53%) and falls (26%). Mean Glasgow coma scale (GCS) score was 4.7. At 6-24 months, quality MRI and functional outcome data were available for 213 and 104 patients, respectively. The best single-area predictors of early and late long-term functional outcome were the ventral midbrain (AUC6mo 0.65 (95% CI 0.56-0.74), p = 0.010) and dorsal pons (AUC24mo 0.68 (95% CI 0.51-0.86) p = 0.001). The best multiple-lesion predictors of poor long-term functional outcome (GOSE < 5) were dorsal pons and splenium of corpus callosum (AUC24mo 0.92 (95% CI 0.84-0.99), p < 0.0001) and the dorsal and ventral pons (AUC24mo 0.91 (95% CI 0.79-1.00), p < 0.0001). The strength of these combinations’ predictive value was not improved by considering three or more areas. The 6-area cluster of internal capsule, corpus callosum genu, body, and splenium; and dorsal and ventral midbrain lesions was a good predictor of outcome at 24 months (AUC24mo 0.82 (95% CI 0.66-0.98), p < 0.0001). CONCLUSIONS: In patients with severe TBI, the strongest predictors of poor long-term functional outcome were combined lesions in the splenium and dorsal pons and the dorsal and ventral pons.

Contemporary Management of Pediatric Brainstem Tumors.

Brain tumors are the second most common malignancy in childhood. Around 15-20% of pediatric brain tumors occur in the brainstem. The most common type of brainstem tumor are diffuse tumors in the ventral pons, whereas focal tumors tend to arise from the midbrain, medulla, and dorsal pons. Glioma is the most common pathological entity. Contemporary management consists of surgery, radiotherapy, chemotherapy, and other adjuvant treatment. Surgical options range from biopsy to radical excision. Biopsy can be performed for diagnostic and prognostic purposes, or in the setting of clinical trials, mainly for diffuse intrinsic pontine gliomas. For focal tumors, surgeons need to carefully balance clinical outcomes against possible neurological sequelae in order to achieve maximal safe resection. Radiotherapy is essential for control of high-grade tumors and may be applied to residual or recurrent low-grade tumors. Proton therapy may provide similar efficacy and less neurotoxicity in comparison to conventional photon therapy. Oncological treatment continues to evolve from conventional chemotherapy to targeted therapy, immunotherapy, and other novel treatment methods and holds great potential as adjuvant therapy for pediatric brainstem tumors.

Medial Pontine Area: A Safe Entry to the Brainstem as a Cut Above the Rest.

To examine the fiber-based anatomy of the medial pontine area (MPA), one of the most commonly used brainstem (BS) safe entry zones in neurosurgery. According to the protocol of Klingler and Ludwig, six BSs were kept in 10% formalin solution for at least 2 months. After removing the arachnoid mater, pia mater, and vascular structures, the samples were frozen at -16°C for at least 2 weeks. White matter (WM) pathways of the BS were gradually examined using fiber dissections under a surgical microscope. Safe entry zones of the BS were defined and investigated, focusing on the ventral pontine region and pontomesencephalic junction. Because of the lack of fibers on the anterior surface of the pons, the MPA formed a safe surgical area. The MPA, strategically positioned between the descending corticospinal tracts and extending securely to the anterior limit of the medial lemniscus, serves as a protective pathway, creating a secure environment for accessing safe entry zones within the BS during surgery. The position of the MPA has the potential to provide a combined surgical path with superiorly located BS entry zones, resulting in a larger surgical area. Entry to the BS via the MPA increases the accessible surface area in the ventral pons and can be combined with the other perioculomotor safe regions outlined. Our findings might lead to safer endoscopic endonasal transclival interventions for intrinsic pontine lesions.

Delayed Neurotoxic Paralysis with Confounding Hypokalaemia

Snakebite is a major health issue in India and developing nations across the globe. Common krait bites are associated with delayed paralysis and locked-in syndrome. Locked-in syndrome has been documented among patients of snakebite. Our case is unique due to the confounding effect of hypokalaemia which may confuse the physician as hypokalaemic periodic paralysis (HPP), a rare genetic condition which can also be precipitated by a stressful event such as snakebite. To the best of our knowledge, we could not associate the presence of hypokalaemia in patients of neurotoxic snakebite. As krait bite are often not associated with the history of having witnessed, a snake due to its nocturnal habit and painless bite, such hypokalaemia should not lead the physician to misdiagnose this snakebite as severe HPP. We urge our fellow researchers to look out for similar findings in their patients. Key messages: Locked-in syndrome in snakebites can be due to the effect of the venom in blood onto the ventral pons. To the best of our knowledge, we could not associate the presence of hypokalaemia in patients of neurotoxic snakebite. We presume this hypokalaemia to be nutritional in origin. As krait bites are often not associated with the history of having witnessed, a snake due to its nocturnal habit and painless bite, such hypokalaemia should not lead the physician to misdiagnose this snakebite as severe HPP. Keywords: Case report, Hypokalaemia, Neurotoxic, Snake envenomation

Resting-state functional connectivity in a non-human primate model of cortical ischemic stroke in area F1

BackgroundThe application of functional MRI to non-human primates after stroke has not yet been undertaken. This is the first study to explore the functional connectivity changes in non-human primate models during acute stages after stroke onset. MethodsNineteen healthy male cynomolgus monkeys (4–5 years) were used in this study. The photothrombosis model was employed to induce focal ischemic stroke in F1 area in the monkey's left hemisphere. T1-weighted structural images and resting-state functional magnetic resonance imaging (rs-fMRI) of all subjects were obtained using a 3.0 Tesla MRI system on the third day following stroke. Based on the D99 atlas, the structural and functional changes of bilateral F1 areas in monkeys were analyzed using region of interest (ROI)-based functional connectivity (FC). The bilateral F1 areas were selected as the seed regions due to their crucial role in motor control and their potential to unveil the comprehensive functional reorganization of the motor system at a whole-brain level following stroke. ResultsIschemic lesions were observed after the stroke, with larger lesion volumes associated with poorer neurological dysfunction. Compared with baseline condition, left area F1 demonstrated decreased FC with the left cerebellum, left ventral pons and left 5_(PEa). When the ROI was located in the right area F1, ischemic monkeys showed decreased FC in left ventral pons, left cerebellum, left primary visual cortex and left 5_(PEa), accompanied by increased FC in the right orbitofrontal cortex. Importantly, the degree of altered FC between left area F1 and left cerebellum was associated with upper limb tone. ConclusionsThese results provide valuable insights into the early-stage functional connectivity changes in the F1 areas of monkeys under ischemic conditions, highlighting the potential involvement of specific brain regions in the pathophysiology of ischemic injury.

XRT-02. SINGLE-CELL RNA SEQUENCING IDENTIFIES FUNCTIONAL MACROPHAGE SUBSETS THAT ARE ENRICHED IN RESPONSE TO DIFFERENTIAL PHAGOCYTOSIS INDUCTION AGAINST GLIOMA

Abstract Pediatric diffuse midline gliomas (DMGs), are fatal brain tumors of childhood arising in the ventral pons. Currently, radiation therapy (RT) is the mainstay treatment for DIPG. However, RT is not a curative treatment and provides only temporary relief for most patients. Recent advances in immunotherapy have yielded some fantastic opportunities to effectively treat patients with high-grade pediatric brain tumors. In this study, we demonstrate that fractionated RT (4Gy X 3) induces immunogenic cell death and activates multiple damage-associated molecular patterns (DAMPs) on DMG/DIPG cells. Furthermore, combining 4Gy X 3 with anti-CD47 therapy enhances the in vitro phagocytosis of DIPG/DMG cells by peripheral blood mononuclear cell-derived macrophages. Next, using single-cell RNA sequencing (scRNA-seq), we investigated the transcriptomic profile of macrophages that were co-cultured with irradiated or non-irradiated DMG cells in the presence of anti-CD47 monoclonal antibodies for 24 hours. Our findings identified eleven distinct macrophage clusters displaying different gene expression patterns in all the treatment conditions. However, PBS treatment led to a marked increase in macrophages expressing antigen-presentation genes (HLA-DR and HLA-DQB1). 4Gy X 3 treatment led to the enrichment of macrophages expressing genes related to the oxidative phosphorylation pathway, whereas anti-CD47 treatment led to the enrichment of macrophages expressing genes related to interferon-gamma response and ERK-signaling pathway. Lastly, mice intracranially transplanted with DMG/DIPG that received 4Gy X 3 and anti-CD47 therapy showed a significant decrease in tumor growth and an increase in survival rate than those receiving either monotherapy alone. We are currently performing scRNA-seq validation studies on tumors obtained from orthotopic and syngeneic models of DMG. In summary, our results highlight the functional heterogeneity of macrophages and suggest that combining fractionated RT with anti-CD47 therapy has distinct anti-tumor effects and can be used as a therapeutic approach for treating DIPG patients.

Comparison of the Retrosigmoid Suprameatal and Anterior Subtemporal Transpetrosal Approaches After Full Exposure of the Internal Acoustic Meatus.

Surgical approaches to the ventral brainstem and petroclival regions are complex, and standard retrosigmoid and subtemporal approaches are often inadequate. Retrosigmoid suprameatal tubercle (RSMTA) and anterior subtemporal transpetrosal (ASTA) approaches may provide extended surgical exposure with less brain retraction. The objective of this study was to evaluate advantages and disadvantages of RSMTA vs ASTA, and illustrate the surgical corridors and 3-dimensional microsurgical anatomy of the related structures. Four silicone-injected adult cadaver heads (8 sides) were dissected to evaluate the accessibility of lesions located at the petrous apex, ventral brainstem, and pontomedullary region using ASTA and RSTMA. Both ASTA and RSMTA provide access from the petrous apex to the ventral lower pons and pontomedullary junction. A greater extent of safely resected bone was found in ASTA vs RSMTA. ASTA provides a larger surgical view to the ventrolateral midpons, peritrigeminal region, superior neurovascular complex, pontomesencephalic junction, and posterior cavernous sinus. Meanwhile, through cranial nerve V mobilization, RSMTA provides a larger surgical view to the lower half part of the pons, ventrolateral part of the pontomedullary junction, and middle and lower neurovascular structures. The choice of surgical approach is determined by considering the area where the lesion originates, lesion size, the anatomic structures to which it extends, and evaluation of the area that can be surgically exposed. Our study highlights the differences between these approaches and important surgical anatomic considerations.

Locked in Syndrome After Penetrating Traumatic Brain Injury

We report a case of locked in syndrome after direct injury to the ventral pons following penetrating trauma. Locked in syndrome is a devastating and rare neurologic disorder that most commonly occurs after cerebrovascular accident involving the basilar artery. Traumatic etiology is rare but in previously reported cases has involved blunt vascular injury as the immediate cause. We present a case of a young male who suffered a penetrating wound to the pons that resulted in locked in syndrome. The diagnosis was confirmed by thorough physical examination, CT and MR imaging, and digital subtraction angiography. Locked in syndrome is an exceptionally rare entity in trauma. Prompt diagnosis requires careful physical examination and high clinical suspicion after appropriate mechanism of injury.

A Case of Brainstem Cryptococcoma Causing Pontine Stroke in an Immunocompetent Male (P1-10.003)

<h3>Objective:</h3> N/A <h3>Background:</h3> Cryptococcus is an invasive fungal disease seen in immune compromised individuals and usually present as meningitis when involved in the Central Nervous System. Cerebral cryptococcoma is a rare form of cryptococcal infection which is often misdiagnosed. Literature reviews showed most of them are located in frontal lobe, parietal lobe or basal ganglia. The involvement of pons only has been extremely rare. There are cases of cryptococcal meningitis causing ischemic stroke due to vasculitis but this case was complicated by perforator infarct adjacent to the lesion. <h3>Design/Methods:</h3> N/A <h3>Results:</h3> <h3>Case presentation:</h3> We are going to discuss a 58 years old immunocompetent male who initially presented with dizziness and double vision. MRI of brain showed small acute infarct in right paramedian pons with adjacent edema out of proportion to the infarct. The contrast showed ring enhancing lesion. The patient returned after 4 months with severe headache and double vision. Repeat MRI of brain showed slightly enlarged ring enhancing lesion in right ventral pons. CSF study was positive for Cryptococcus neoformans both times. <h3>Conclusions:</h3> Diagnosis of cryptococcoma is often challenging especially in immunocompetent individuals where the clinical suspicion is low. There have been case reports where it has been misdiagnosed as neoplasm, tumefactive demyelinating disease and stroke. Therefore, causing delays in treatment leading to increased mortality and morbidity. This discussion indicates that physicians should have low threshold to suspect cryptococcoma in immunocompetent individual with ring enhancing lesion as timely intervention can save lives from this invasive fungal disease. <b>Disclosure:</b> Dr. Jabeen has nothing to disclose. Dr. Maghsoudlou has nothing to disclose.

Understanding the Psychological Well-being of Patients With Locked-in Syndrome: A Scoping Review.

Locked-in syndrome (LiS) is a neurological disorder caused by lesions affecting the ventral pons and midbrain and is characterized by loss of physical function, but with perceived consciousness intact. Despite severely limited function, previous studies have shown the quality of life (QoL) in patients to be more positive than naturally assumed by caregivers and relatives. The present review aims to synthesize the broad scientific literature focused on the psychological well-being of LiS patients. A scoping review was performed to synthesize the available evidence on the psychological well-being of LiS patients. Eligible studies included those that target individuals with LiS as the study population, evaluated psychological well-being, and explored the factors related to it. We extracted study population details, type of QoL methods, method of communication, and primary findings from the studies. We summarized the findings categorized into health-related QoL (HRQoL), global QoL, and other tools for assessing psychological status. Across the 13 eligible studies, weobserved that patients with LiS had reasonable or similar psychological well-being as the standard based on HRQoL and global QoL assessment. Caregivers and healthcare professionals seem to rate the psychological QoL of LiS patients lower than patients themselves. Studies showed evidence that the longer duration of LiS is a factor that positively affects QoL, and augmentative and alternative communication tools and recovery of speech production showed positive effects as well. Studies reported a range of 27% to 68% of patients experiencing thoughts of suicide and euthanasia. The evidence shows that LiS patients had reasonable psychological well-being. There appear to be differences between patients' assessed well-being and the negative perceptions by caregivers. Response shift and adaptation to disease by patients are considered potential reasons. A sufficient moratorium period and provision of information to support patients' QoL and appropriate decision-making seems necessary.

Pediatric diffuse midline glioma: Understanding the mechanisms and assessing the next generation of personalized therapeutics.

Diffuse midline glioma (DMG) is a pediatric cancer that originates in the midline structures of the brain. Prognosis of DMG patients remains poor due to the infiltrative nature of these tumors and the protection they receive from systemically delivered therapeutics via an intact blood-brain barrier (BBB), making treatment difficult. While the cell of origin remains disputed, it is believed to reside in the ventral pons. Recent research has pointed toward epigenetic dysregulation inducing an OPC-like transcriptomic signature in DMG cells. This epigenetic dysregulation is typically caused by a mutation (K27M) in one of two histone genes-H3F3A or HIST1H3B -and can lead to a differentiation block that increases these cells oncogenic potential. Standard treatment with radiation is not sufficient at overcoming the aggressivity of this cancer and only confers a survival benefit of a few months, and thus, discovery of new therapeutics is of utmost importance. In this review, we discuss the cell of origin of DMGs, as well as the underlying molecular mechanisms that contribute to their aggressivity and resistance to treatment. Additionally, we outline the current standard of care for DMG patients and the potential future therapeutics for this cancer that are currently being tested in preclinical and clinical trials.

Selective vulnerability of brainstem and cervical spinal cord regions in people with non-progressive multiple sclerosis of Black or African American and European ancestry

Background: We evaluated imaging features suggestive of neurodegeneration within the brainstem and upper cervical spinal cord (UCSC) in non-progressive multiple sclerosis (MS). Methods: Standardized 3-Tesla three-dimensional brain magnetic resonance imaging (MRI) studies were prospectively acquired. Rates of change in volume, surface texture, curvature were quantified at the pons and medulla-UCSC. Whole and regional brain volumes and T2-weighted lesion volumes were also quantified. Independent regression models were constructed to evaluate differences between those of Black or African ancestry (B/AA) and European ancestry (EA) with non-progressive MS. Results: 209 people with MS (pwMS) having at least two MRI studies, 29% possessing 3–6 timepoints, resulted in 487 scans for analysis. Median follow-up time between MRI timepoints was 1.33 (25th–75th percentile: 0.51–1.98) years. Of 183 non-progressive pwMS, 88 and 95 self-reported being B/AA and EA, respectively. Non-progressive pwMS demonstrated greater rates of decline in pontine volume (p < 0.0001) in B/AA and in medulla-UCSC volume (p < 0.0001) for EA pwMS. Longitudinal surface texture and curvature changes suggesting reduced tissue integrity were observed at the ventral medulla-UCSC (p < 0.001), dorsal pons (p < 0.0001) and dorsal medulla (p < 0.0001) but not the ventral pons (p = 0.92) between groups. Conclusions: Selectively vulnerable regions within the brainstem-UCSC may allow for more personalized approaches to disease surveillance and management.

IMMU-04. SINGLE-CELL RNA-SEQUENCING IDENTIFIES FUNCTIONAL MACROPHAGE SUBSETS THAT ARE ENRICHED IN RESPONSE TO DIFFERENTIAL PHAGOCYTOSIS INDUCTION AGAINST GLIOMA

Abstract Pediatric diffuse midline gliomas (DMGs), are fatal brain tumors of childhood arising in the ventral pons. Currently, radiation therapy (RT) is the mainstay treatment for DIPG. However, RT is not a curative treatment and provides only temporary relief for most patients. Recent advances in immunotherapy have yielded some fantastic opportunities to effectively treat patients with high-grade pediatric brain tumors. In this study, we demonstrate that fractionated RT (4Gy X 3) induces immunogenic cell death and activates multiple damage-associated molecular patterns (DAMPs) on DMG/DIPG cells. Furthermore, combining 4Gy X 3 with anti-CD47 therapy enhances the in vitro phagocytosis of DIPG/DMG cells by peripheral blood mononuclear cell-derived macrophages. Next, using single-cell RNA sequencing (scRNA-seq), we investigated the transcriptomic profile of macrophages that were co-cultured with irradiated or non-irradiated DMG cells in the presence of either phosphate-buffered solution (PBS) or anti-CD47 monoclonal antibody for 24 hours. Our findings identified eleven distinct macrophage clusters displaying different gene expression patterns in all the treatment conditions. However, PBS treatment led to a marked increase in macrophages expressing antigen-presentation genes (HLA-DR and HLA-DQB1). 4Gy X 3 treatment led to the enrichment of macrophages expressing genes related to the oxidative phosphorylation pathway, whereas anti-CD47 treatment led to the enrichment of macrophages expressing genes related to interferon-gamma response and ERK-signaling pathway. Lastly, mice intracranially transplanted with DMG/DIPG that received 4Gy X 3 and anti-CD47 therapy showed a significant decrease in tumor growth and an increase in survival rate than those receiving either monotherapy alone. We are currently performing scRNA-seq validation studies on tumors obtained from orthotopic and syngeneic models of DMG. In summary, our results highlight the functional heterogeneity of macrophages and suggest that combining fractionated RT with anti-CD47 therapy has potent anti-tumor effects and may be used as a novel therapeutic approach for treating Glioma patients.

A CASE REPORT ON: BASILAR ARTERY ANEURYSM WITH CANCER

Introduction: Basilar artery provide oxygenated blood to the brain. The vertebral basilar system, which supplies blood to the posterior region of the Willis circle and joins with blood provided to the anterior half of the Willis process from the internal carotid arteries, is made up of the two vertebral arteries and the basilar artery. At the medulla oblongata and pons of the abducens nerves, the two vertebral arteries merge to form the basilar artery. The basilar artery's diameter ranges from 1.5 to 6.6 mm. When the midbrain ascends superiorly in the basilar sulcus of the ventral pons and separates into the posterior cerebral arteries, the midbrain and pons come together. Present complaint and investigation: A-53-year-old female was hospitalized in rural hospital Wardha with complaints of breathing difficulty, lower limb swelling, nausea, and vomiting and chest pain, mucus in the throat and lungs, and loss of appetite since 12 days. Investigation: Haemoglobin is decreased by 10.7gm, WBC count 11,000 per microliter, Total R.B.C. count increased by 5.84 million /cell, M.C.H. level Decreased 70fl, MCH is decreased by 22.8 Pico-gm. Therefore CT report should be interpreted in correlation with clinical &amp; pathological findings. Past History: He had been admitted eight days before outside the hospital for the same complaints. The patient had complaints of Intraventricular Haemorrhage on treatment after investigations and the physical examination diagnosed basilar artery aneurysm with cancer. Conclusion: The prognosis of disease severity can be fair by timely treatment and management of cancer and basilar artery aneurysm.

Abstract 5716: Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG)

Abstract DIPG is an incurable and inoperable pediatric brain cancer in the ventral pons characterized by its complex mutational profile. Epigenetic mutations to H3K27M are seen in up to 81% of patients, resulting in the global loss of the repressive H3K27 trimethylation marker, increasing chromatin accessibility and deregulating gene expression. However, these epigenetic changes are often partnered by secondary mutations, with activating mutations and copy number gains observed in a wide variety of RTK genes and their associated signaling pathways. The combination of these two mutational paradigms results in the broad alteration of cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper, a metal ion essential for normal cellular function is known to be highly expressed in the pons, and influence ERK and PI3K/AKT signaling. Importantly, excess copper accumulation has been implicated in the pathogenesis of multiple neurological diseases and cancers. Copper chelation therapy is a clinically approved strategy for pediatric patients with Wilson’s Disease, known to improve their neurological symptoms, and is under investigation for use in a number of cancers. Therefore, we hypothesized copper chelation may be an effective therapeutic strategy for DIPG. Cytotoxicity assays using copper chelator tetraethylenepentamine (TEPA) demonstrated millimolar efficacy and synergized with mTOR inhibitor everolimus in a panel of DIPG cell lines, with H3-K27M mutant cell lines found to be more sensitive. Western blots were used to identify copper integrated pathways, with DIPG cell lines stimulated with sub-lethal copper concentrations demonstrating increased phosphorylated expression of ERK1/2, ERK5, AKT and p70SK61. TEPA reduced the phosphorylation of these markers compared to controls. Importantly, epigenetic mechanisms were sensitive to copper, as both copper stimulation and TEPA modulated H3K27 trimethylation, and TEPA decreased pro-expression H3K27 acetylation. In further blots examining epigenetic regulators, TEPA reduced the expression of EZH2, DNMT3B and DNMT1. RNA-sequencing analysis revealed TEPA downregulated genes involved in S-adenosylmethionine production metabolism, DNA regulation, cell cycle and mTORC signaling and specifically, EZH2, DNMT3B and DNMT1 expression. In vivo investigations demonstrated TEPA’s ability to improve survival in an orthotopic xenograft model, and via xenogen luminescence imaging complete tumor regression in 25% of treated mice. This study indicates copper directly impacts epigenetic and RTK mechanisms, and that their targeting through copper chelation agents represents a potential therapeutic strategy for DIPG, both as a single agent and in combination. Citation Format: Filip Michniewicz, Federica Saletta, Tayla Watkinson, Daniele Mercatelli, Federico M. Giorgi, Jessica Bell, Maria Tsoli, David Ziegler, Orazio Vittorio. Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5716.

DIPG-20. Copper chelation therapy targets S-adenosylmethionine (SAM) metabolism and epigenetic regulators in diffuse intrinsic pontine glioma (DIPG)

DIPG is an incurable pediatric brain cancer of the ventral pons characterized by its complex epigenetic profile. Up to 81% of patients present with mutation H3K27M, resulting in the global reduction of H3K27 trimethylation and increased H3K27 acetylation, deregulating gene expression through an aberrant pattern of epigenetic modification. These alterations affect many cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper is highly abundant in the pons and is essential for normal brain function and development. However, excess copper accumulation is implicated in several neurological diseases and cancers. Incidentally, recent investigations have indicated the H3-H4 dimer can interact with copper acting as a reductase enzyme. Copper chelation therapy is clinically approved for pediatric patients with Wilson’s Disease, improving their neurological symptoms, and is being trialed in several cancers. We therefore hypothesized copper chelation may represent an effective therapeutic strategy for DIPG. Copper chelator tetraethylenepentamine (TEPA) decreased cell growth and induced apoptosis in DIPG cell lines. To understand these results, unbiased RNA-seq and metabolomics analyses were performed, revealing downregulation of EZH2, DNMT1 and DNMT3B, upregulation of KDM6B and the disruption of key enzymes in the S-adenosylmethionine (SAM)-cycle. Importantly, TEPA downregulated SAM, which donates methyl groups for methylation, S-adenosylhomocysteine, its post-methylation product and α-ketoglutarate, a co-factor for KDM6B. Western blots confirmed the reduced expression of EZH2, DNMT1 and DNMT3B, with further blots examining the chromatin cell fraction revealing modulation of H3K27 trimethylation through copper or TEPA stimulation, and reduction of H3K27 acetylation by TEPA. Importantly, in vitro combinations with Panobinostat were synergistic, while in vivo investigations demonstrated TEPA improved survival in an orthotopic patient derived xenograft (PDX) model, showing complete tumor regression in 25% of treated mice. This study indicates a novel use for copper chelators as epigenetic drugs, and their potential as therapeutics for DIPG.

A New Case With Cortical Malformation Caused by Biallelic Variants in LAMC3.

ObjectiveIn this study, we report the case of a 24-year-old man with intellectual disability and childhood-onset seizures. This patient had newly identified biallelic variants in the laminin subunit gamma 3 (LAMC3) gene with unreported cortical malformation.MethodsExome sequencing.ResultsGenetic analyses revealed new biallelic variants in the LAMC3 gene. An MRI examination of the brain revealed cortical malformations predominantly in the temporal lobes and mildly in the occipital, frontal, and parietal lobes. In addition, our patient also exhibited mild midline malformation in the ventral pons, which is unique to LAMC3 variants.DiscussionPatients with LAMC3 variants have been reported to exhibit cortical malformation predominantly in the occipital lobes, but this patient exhibited cortical malformation predominantly in the temporal lobes and mildly in the occipital, frontal, and parietal lobes. In addition, this patient also exhibited mild midline malformation in the ventral pons. These unique findings cast new light on the role of LAMC3 in brain development.