A novel combination approach to effectively reduce inflammation and neurodegeneration in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated attacks on the central nervous system (CNS), resulting in demyelination and recurring T-cell responses. Unfortunately, there is no cure for it. Current therapies that target immunomodulation and/or immunosuppression show only modest beneficial effects, have many side effects, and do not block neurodegeneration or progression of the disease. Since neurodegeneration and in particular axonal degeneration is implicated in disability in progressive MS, development of novel therapeutic strategies to attenuate the neurodegenerative processes is imperative. This study aims to develop new safe and efficacious treatments that address both the inflammatory and neurodegenerative aspects of MS using its animal model, experimental allergic encephalomyelitis (EAE). In EAE, the cysteine protease calpain is upregulated in CNS tissue, and its activity correlates with neurodegeneration. Our immunologic studies on MS have indicated that increased calpain activity promotes pro-inflammatory T helper (Th)1 cells and the severity of the disease in EAE, suggesting that calpain inhibition could be a novel target to combat neurodegeneration in MS/EAE. While calpain inhibition by SNJ1945 reduced disease severity, treatment of EAE animals with a novel protease-resistant altered small peptide ligand (3aza-APL) that mimic myelin basic protein (MBP), also decreased the incidence of EAE, disease severity, infiltration of inflammatory cells, and protected myelin. A reduction in inflammatory T-cells with an increase in Tregs and myeloid suppressor cells is also found in EAE mice treated with SNJ1945 and 3aza-APL. Thus, a novel combination strategy was tested in chronic EAE mouse model in B10 mice which showed multiple pathological mechanisms could be addressed by simultaneous treatment with calpain inhibitor SNJ1945 and protease-resistant 3aza-APL to achieve a stronger therapeutic effect.

Case report: Awake craniotomy during pregnancy for resection of glioblastoma.

Few cases have been reported of the diagnosis and treatment of glioblastoma (GB) during pregnancy. Subsequently, surgical, medical, and obstetrical management of complicated primary central nervous system malignancy in antepartum and postpartum patients remains under-investigated. The authors report the case of a 24-year-old female patient who developed generalized tonic-clonic seizures and focal neurologic deficits. MRI imaging (3T Skyra, Siemens, Erlangen, Germany) revealed an intracranial mass suspicious for malignant tumor and surgical resection under awake sedation was scheduled. The patient was incidentally found to be in her first trimester of pregnancy. Using neuronavigation, neurophysiologic monitoring, and conscious sedation the tumor was debulked successfully and histopathologic analysis confirmed giant cell glioblastoma, WHO Grade IV, 1p/19q intact, IDH wild-type, with NF1 p.Y2285fs and RB1 p.S318fs somatic mutations. Post-surgical oncologic management continued with fractioned radiotherapy and use of the Optune® device. The patient underwent uncomplicated cesarean section at 34-weeks gestation, the child remains healthy and the patient remains disease-disease free at 1-year. Thus, this case presents an approach to management of complicated GBM during first trimester pregnancy.

Premarin Reduces Neurodegeneration and Promotes Improvement of Function in an Animal Model of Spinal Cord Injury.

Spinal cord injury (SCI) causes significant mortality and morbidity. Currently, no FDA-approved pharmacotherapy is available for treating SCI. Previously, low doses of estrogen (17ß-estradiol, E2) were shown to improve the post-injury outcome in a rat SCI model. However, the range of associated side effects makes advocating its therapeutic use difficult. Therefore, this study aimed at investigating the therapeutic efficacy of Premarin (PRM) in SCI. PRM is an FDA-approved E2 (10%) formulation, which is used for hormone replacement therapy with minimal risk of serious side effects. The effects of PRM on SCI were examined by magnetic resonance imaging, immunofluorescent staining, and western blot analysis in a rat model. SCI animals treated with vehicle alone, PRM, E2 receptor antagonist (ICI), or PRM + ICI were graded in a blinded way for locomotor function by using the Basso-Beattie-Bresnahan (BBB) locomotor scale. PRM treatment for 7 days decreased post-SCI lesion volume and attenuated neuronal cell death, inflammation, and axonal damage. PRM also altered the balance of pro- and anti-apoptotic proteins in favor of cell survival and improved angiogenesis and microvascular growth. Increased expression of estrogen receptors (ERs) ERα and ERß following PRM treatment and their inhibition by ER inhibitor indicated that the neuroprotection associated with PRM treatment might be E2-receptor mediated. The attenuation of glial activation with decreased inflammation and cell death, and increased angiogenesis by PRM led to improved functional outcome as determined by the BBB locomotor scale. These results suggest that PRM treatment has significant therapeutic implications for the improvement of post-SCI outcome.

Establishing a Standardized Method for the Effective Intraoperative Collection and Biological Preservation of Brain Tumor Tissue Samples Using a Novel Tissue Preservation System: A Pilot Study.

BACKGROUND: Glioblastoma (GB) is an aggressive tumor showing extensive intertumoral and intratumoral heterogeneity. Several possible reasons contribute to the historical inability to develop effective therapeutic strategies for treatment of GB. One such challenge is the inability to consistently procure high-quality biologically preserved specimens for use in molecular research and patient-derived xenograft model development. No scientifically derived standardized method exists for intraoperative tissue collection specifically designed with the fragility of RNA in mind. METHODS: In this investigation, we set out to characterize matched specimens from 6 GB patients comparing the traditional handling and collection processes of intraoperative tissue used in most neurosurgical operating rooms versus an automated resection, collection, and biological preservation system (APS) which captures, preserves, and biologically maintains tissue in a prescribed and controlled microenvironment. Matched specimens were processed in parallel at various time points and temperatures, evaluating viability, RNA and protein concentrations, and isolation of GB cell lines. RESULTS: We found that APS-derived GB slices stored in an APS modified medium remained viable and maintained high-quality RNA and protein concentration for up to 24 hours. CONCLUSIONS: Our results showed that primary GB cell cultures derived in this manner had improved growth over widely used collection and preservation methods. By implementing an automated intraoperative system, we also eliminated inconsistencies in methodology of tissue collection, handling and biological preservation, establishing a repeatable and standardized practice that does not require additional staff or a laboratory technician to manage it.

Analysis of the P53N a Novel Protein Encoded on Chromosome 22q12.1-12.3 in Glioblastomas and Ependymomas Specimens.

The P53N gene maps precisely to human chromosome sub-band 22q12.1-12.3, a region where loss of heterozygosity has been reported in 30% of astrocytic tumors and associated with progression to anaplasia. Moreover, a putative tumor suppressor gene has been indicated on 22q11 region involved in pathogenesis of ependymal tumors. Our objectives to examine the expression level of novel membrane-associated protein (termed P53N) encoded by a novel human gene on chromosome 22q12.1-12.3 in glioblastomas and ependymomas. Serial analysis of gene expression (SAGE) and immunofluorescence analysis of the P53N in the brain tumor tissues were performed. Our analysis revealed that there was high expression of the P53N mRNA in brain ependymoma and brain well-differentiated astrocytoma libraries. The P53N protein. P53N protein contains a high mobility group (HMG) domain at amino acid positions 301 to 360 expressed highly in glioblastoma and ependymoma specimens. Anti-P53N carboxyl-terminal peptide antibody localized the P53N protein to the cytoplasmic membranes of protoplasmic astrocytes in the glioblastoma and ependymoma specimens. These results are in good agreement with the SAGE analysis and the predicted transmembrane topology for the P53N protein and support a possible transmembrane model in which the P53N contains a predicted transmembrane region with its amino terminus localized to the inside of the cytoplasmic membrane.

Preconditioning with INC280 and LDK378 drugs sensitizes MGMT-unmethylated glioblastoma to temozolomide: Pre-clinical assessment.

Temozolomide (TMZ) therapy is the standard of care for patients with glioblastoma (GBM). Clinical studies have shown that elevated levels of DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT) or deficiency/defect of DNA mismatch repair (MMR) genes is associated with TMZ resistance in some, but not all, GBM tumors. Another reason for GBM treatment failure is signal redundancy due to coactivation of several functionally linked receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK) and c-Met (hepatocyte growth factor receptor). As such, these tyrosine kinases serve as potential targets for GBM therapy. Thus, we tested two novel drugs: INC280 (Capmatinib: a highly selective c-Met receptor tyrosine kinase-RTK inhibitor) and LDK378 (Ceritinib: a highly selective anaplastic lymphoma kinase-ALK inhibitor), aiming to overcome TMZ resistance in MGMT-unmethylated GBM cells in in vitro cell culture models. Treatments were examined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, caspase-3 assay and western blot analysis. Results obtained from our experiments demonstrated that preconditioning with INC280 and LDK378 drugs exhibit increased MMR protein expression, specifically MMR protein MLH1 (MutL Homolog 1) and MSH6 (MutS Homolog 6) and sensitized TMZ in MGMT-unmethylated GBM cells via suppression of ALK and c-Met expression. INC280 and LDK378 plus TMZ also induced apoptosis by modulating downstream signaling of PI3K/AKT/STAT3. Taken together, this data indicates that co-inhibition of ALK and c-MET can enhance growth inhibitory effects in MGMT-unmethylated cells and enhance TMZ sensitivity in-vitro, suggesting c-Met inhibitors combined with ALK-targeting provide a therapeutic benefit in MGMT-unmethylated GBM patients.

Targeting Subventricular Zone Progenitor Cells with Intraventricular Liposomal Encapsulated Cytarabine in Patients with Secondary Glioblastoma : A Report of Two Cases.

BACKGROUND: Current treatments for glioblastoma (GB), the most common and malignant primary brain tumor are inadequate and as such, the median survival for most patients with GB is on the order of months, even after cytoreductive surgery, radiation and chemotherapy. CASE DESCRIPTION: Current study reports two cases of glioblastoma (GB) with subventricular zone (SVZ) involvement. SVZ biopsies demonstrated the presence of hypercellularity, nestin immunoreactivity, and a Ki-67 labeling index (LI) of 1-2%. Interestingly, tumor morphology and proliferative indices are different in the SVZ specimens than the hemispheric recurrences, which displayed similar nestin immunoreactivity, but a greater LI of 10%. Biopsy specimens demonstrated both intense nestin immunoreactivity and GFAP immunoreactivity in and around the GB recurrence. Nestin positive cells were more abundant closer to the SVZ nearest to the dorsolateral horn of the left lateral ventricle, while GFAP immunoreactivity was more intense closer to the center of the tumor recurrence. Additionally, co-labeling of cells with Ki67 and several different progenitor markers (CD133, CD140, TUJ-1, and nestin) demonstrated that these cells found in and around the GB recurrence were actively dividing. Having failed standard therapy with evidence of bi-hemispheric spread and progression to GB, we report a novel approach of using intraventricular liposomal encapsulated cytarabine (DepoCyt) for the treatment for GB by suppressing glial progenitor cells that surround the ventricular system in patients with GB. CONCLUSIONS: MRI and immunohistochemistry demonstrated that the SVZ is the incubator for future recurrences of GB and propose targeting SVZ progenitor cells with intraventricular liposomal encapsulated Ara-C. Two patients treated using this novel regimen have demonstrated partial radiographic responses warranting further studies looking at targeting the subventricular zone.

Analysis of P78: A Novel Cytoplasmic Membrane-Associated Protein Encoded on Chromosome 19q13.3 in Glioma Specimens.

Allelic losses of the q13.3 region of chromosome 19 have been documented in all major types of diffuse gliomas, strongly suggesting the presence of a 19q13.3 tumor suppressor gene responsible for these malignancies. The P78 gene precisely maps to 19q13.3, the glioma candidate region, and encodes a recently identified novel protein (P78). The purpose of this study was to determine P78 protein expression in gliomas. Serial analysis of gene expression (SAGE) reveals P78 mRNA expression to be significantly reduced in high-grade gliomas such as glioblastoma (GB), as compared with the low-grade tumors including astrocytomas, oligodendrogliomas, and ependymomas. We observed the distribution of staining of P78 protein was concentrated on the cell membranes of the luminal epithelial cells, not cytoplasm. In contrast, the pre-immune serum controls demonstrated no staining. These results demonstrate that P78 protein is highly expressed in the cytoplasmic membranes of low but not high-grade astrocytomas, and correlates with grade of malignancy. In these double immunostaining experiments, the anti-Map-2 and anti-NeuN antibodies did not stain round cells that were stained with the anti-P78 carboxyl-terminal peptide antibodies, demonstrating that these round cells were not neurons, and likely protoplasmic astrocytes. Current results also suggest that the astrocytes stained with the anti-P78 carboxyl-terminal peptide antibody are likely protoplasmic astrocytes. We also observed preincubation of anti-P78 carboxyl-terminal antibodies with immunizing peptides abolished immunostaining in gliomas. These results suggest a role for the P78 protein in the process of abnormal growth in glial tumors.

In Vitro Effect of Dovitinib (TKI258), a Multi-Target Angiokinase Inhibitor on Aggressive Meningioma Cells.

Background: Meningiomas represent ∼30% of primary central nervous system (CNS) tumors. Although advances in surgery and radiotherapy have significantly improved survival, there remains an important subset of patients whose tumors have more aggressive behavior and are refractory to conventional therapy. Recent advances in molecular genetics and epigenetics suggest that this aggressive behavior may be due to the deletion of the DNA repair and tumor suppressor gene, CHEK2, neurofibromatosis Type 2 (NF2) mutation on chromosome 22q12, and genetic abnormalities in multiple RTKs including FGFRs. Management of higher-grade meningiomas, such as anaplastic meningiomas (AM: WHO grade III), is truly challenging and there isn't an established chemotherapy option. We investigate the effect of active multi tyrosine receptor kinase inhibitor Dovitinib at stopping AM cell growth in in vitro with either frequent codeletion or mutated CHEK2 and NF2 gene.Methods: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot analysis, caspases assay, and DNA fragmentation assay.Results: Treatment of CH157MN and IOMM-Lee cells with Dovitinib suppressed multiple angiokinases-mainly FGFRs, leading to suppression of downstream signaling by RAS-RAF-MAPK molecules and PI3K-AKT molecules which are involved in cell proliferation, cell survival, and tumor invasion. Furthermore, Dovitinib induced apoptosis via downregulation of survival proteins (Bcl-XL), and over-expression of apoptotic factors (Bax and caspase-3) regardless of CHEK2 and NF2 mutation status.Conclusions: This study establishes the groundwork for the development of Dovitinib as a therapeutic agent for high-grade AM with either frequent codeletion or mutated CHEK2 and NF2, an avenue with high translational potential.

Tamoxifen is a candidate first-in-class inhibitor of acid ceramidase that reduces amitotic division in polyploid giant cancer cells-Unrecognized players in tumorigenesis.

Polyploid giant cancer cells (PGCC) represent a poorly understood, small subpopulation of tumor cells that are increasingly being recognized for their critical role in therapy resistance, metastasis, and cancer recurrence. PGCC have the potential to generate progeny through primitive or cleavage-like division, which allows them to evade antimitotic insults. We recently demonstrated that the sphingolipid enzyme acid ceramidase (ASAH1) is required for this process. Since specific ASAH1 inhibitors are not clinically available, we investigated whether tamoxifen, which interferes with ASAH1 function via off-target effects, has a potential clinical benefit independent of estrogen signaling. Our results show that tamoxifen inhibits generation of PGCC offspring in prostate cancer, glioblastoma, and melanoma cells. Analysis of two state-level cancer registries revealed that tamoxifen improves survival outcomes for second, nonbreast cancers that develop in women with early stage breast cancer. Our results suggest that tamoxifen may have a clinical benefit in a variety of cancers that is independent of estrogen signaling and could be due to its inhibition of acid ceramidase. Thus the distinct application of tamoxifen as potentially a first-in-class therapeutic that inhibits the generation of PGCC offspring should be considered in future clinical trials.

Oncolytic myxoma virus synergizes with standard of care for treatment of glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is an aggressive form of brain cancer which is associated with poor prognosis. A variety of oncolytic viruses have previously shown positive efficacy against GBM, potentially offering new treatment options for patients. One such oncolytic virus is Myxoma virus (MYXV), a rabbit-specific poxvirus that has been shown to be efficacious against a variety of tumor models including GBM. PURPOSE: The purpose of this study was to test the efficacy of MYXV combined with current treatment regimens for GBM in both established cell lines as well as patient biopsy samples. MATERIALS AND METHODS: U118 gliobastoma cell lines were treated under various standard of care combinations (untreated, radiation and chemotherapeutic) prior to infection with MYXV. Infection was then monitored for differences in rate of infection, titer and rate of spread. Cellular death was measured by MTT assay and Caspase-3 colorimetric assay. Patient biopsies were harvested and treated under similar treatment conditions. RESULTS: The addition of GBM standard of care to MYXV infection resulted in an increased rate of spread compared to single treatment with either radiation or chemotherapeutic alone. SOC did not alter viral replication or infection rates. Similar effects were seen in ex vivo patient biopsies. Cellular viability was significantly decreased with the combination therapy of SOC and MYXV infection compared to any other treatment outcome. Caspase-3 activity was also significantly increased in samples treated with combination therapy when compared to any other treatment combination. CONCLUSION: Our results show that the combination of MYXV with current SOC results in both increased killing of GBM cells compared to either treatment regime alone as well as increased spread of MYXV infection. These findings lay the foundation for future in vivo studies on combining MYXV with GBM SOC.

Single agent efficacy of the HDAC inhibitor DATS in preclinical models of glioblastoma.

PURPOSE/INTRODUCTION: Glioblastoma (GB) remains incurable despite aggressive chemotherapy, radiotherapy, and surgical interventions; immunotherapies remain experimental in clinical practice. Relevant preclinical models that can accurately predict tumor response to therapy are equally challenging. This study aimed to validate the effect of the naturally occurring agent diallyl trisulfide (DATS) in human GB in relevant pre-clinical models. METHODS: Ex vivo slice culture, in vivo cell line derived orthotopic xenograft and patient-derived orthotopic xenograft (PDX) animal models of GB were utilized to assess efficacy of treatment with DATS. RESULTS: Our results showed 72-h treatments of 25 µM DATS induced cell death in ex vivo human GB slice culture. We treated U87MG orthotopic xenograft models (U87MGOX) and patient-derived orthotopic xenograft models (PDX) with daily intraperitoneal injections of DATS for 14 days. Magnetic resonance (MR) imaging of mice treated with DATS (10 mg/kg) demonstrated reduced tumor size at 5 weeks when compared with saline-treated U87MGOX and PDX controls. Hematoxylin (H&E) staining demonstrated dose-dependent reduction in gross tumor volume with decreased proliferation and decreased angiogenesis. Western blotting showed that DATS was associated with increases in histone acetylation (Ac-Histone H3/H4) and activated caspase-3 in this novel preclinical model. Histological assessment and enzyme assays showed that even the highest dose of DATS did not negatively impact hepatic function. CONCLUSIONS: DATS may be an effective and well-tolerated therapeutic agent in preventing tumor progression and inducing apoptosis in human GB.

Association between skin lesion and arsenic concentration in hair by mixed bivariate model in chronic arsenic exposure.

Skin lesion is one of the important health hazards caused by high intake of arsenic through drinking water and diet, and the other hazards include several types of cancers (viz. skin, lung and urinary bladder), ischemic heart disease, hypertension, etc. Two most important biomarkers to measure arsenic intake in a human body are arsenic concentration in urine and hair. The primary interest of this paper is the association between skin lesion and arsenic concentration in hair for participants with chronic arsenic exposure from West Bengal, India, using bivariate regression model based on copula function. The result showed participants with high arsenic concentration in hair had higher incidence of developing skin lesion. Arsenic concentration in hair was significantly higher for the participants with an arsenic concentration in water > 10 mg/L.

Immunological low-dose radiation modulates the pediatric medulloblastoma antigens and enhances antibody-dependent cellular cytotoxicity.

BACKGROUND: Immunotherapy can be an effective treatment for pediatric medulloblastoma (MB) patients. However, major subpopulations do not respond to immunotherapy, due to the lack of antigenic mutations or the immune-evasive properties of MB cells. Clinical observations suggest that radiation therapy (RT) may expand the therapeutic reach of immunotherapy. The aim of the present investigation is to study the effect of low-dose X-ray radiation (LDXR, 1 Gy) on the functional immunological responses of MB cells (DAOY, D283, and D341). METHODS: Induction of MB cell death was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Production of reactive oxygen species (ROS) was measured by fluorescent probes. Changes in the expression of  human leukocyte antigen (HLA) molecules and caspase-3 activities during treatment were analyzed using Western blotting and caspase-3 assay. RESULTS: Western blot analysis demonstrated that LDXR upregulated the expression of HLA class I and HLA II molecules by more than 20% compared with control and high-dose (12 Gy) groups in vitro. Several of these HLA subtypes, such as MAGE C1, CD137, and ICAM-1, have demonstrated upregulation. In addition, LDXR increases ROS production in association with phosphorylation of NF-κB and cell surface expression of mAb target molecules (HER2 and VEGF). These data suggest that a combined LDXR and mAb therapy can create a synergistic effect in vitro. CONCLUSION: These results suggest that LDXR modulates HLA molecules, leading to alterations in T-cell/tumor-cell interaction and enhancement of T-cell-mediated MB cell death. Also, low-dose radiotherapy combined with monoclonal antibody therapy may one day augment the standard treatment for MB, but more investigation is needed to prove its utility as a new therapeutic combination for MB patients.

Neuron-microglia interaction induced bi-directional cytotoxicity associated with calpain activation.

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Administration of low dose estrogen attenuates persistent inflammation, promotes angiogenesis, and improves locomotor function following chronic spinal cord injury in rats.

Spinal cord injury (SCI) causes loss of neurological function and, depending upon the severity of injury, may lead to paralysis. Currently, no FDA-approved pharmacotherapy is available for SCI. High-dose methylprednisolone is widely used, but this treatment is controversial. We have previously shown that low doses of estrogen reduces inflammation, attenuates cell death, and protects axon and myelin in SCI rats, but its effectiveness in recovery of function is not known. Therefore, the goal of this study was to investigate whether low doses of estrogen in post-SCI would reduce inflammation, protect cells and axons, and improve locomotor function during the chronic phase of injury. Injury (40 g.cm force) was induced at thoracic 10 in young adult male rats. Rats were treated with 10 or 100 µg 17ß-estradiol (estrogen) for 7 days following SCI and compared with vehicle-treated injury and laminectomy (sham) controls. Histology (H&E staining), immunohistofluorescence, Doppler laser technique, and Western blotting were used to monitor tissue integrity, gliosis, blood flow, angiogenesis, the expression of angiogenic factors, axonal degeneration, and locomotor function (Basso, Beattie, and Bresnahan rating) following injury. To assess the progression of recovery, rats were sacrificed at 7, 14, or 42 days post injury. A reduction in glial reactivity, attenuation of axonal and myelin damage, protection of cells, increased expression of angiogenic factors and microvessel growth, and improved locomotor function were found following estrogen treatment compared with vehicle-treated SCI rats. These results suggest that treatment with a very low dose of estrogen has significant therapeutic implications for the improvement of locomotor function in chronic SCI. Experimental studies with low dose estrogen therapy in chronic spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes that could ameliorate the degenerative pathways in chronic SCI as shown in (a). Furthermore, the alterations in local spinal blood flow could be significantly alleviated with low dose estrogen therapy. This therapy led to the preservation of the structural integrity of the spinal cord (b), which in turn led to the improved functional recovery as shown (c).

Molecular mechanisms of estrogen for neuroprotection in spinal cord injury and traumatic brain injury.

Estrogen (EST) is a steroid hormone that exhibits several important physiological roles in the human body. During the last few decades, EST has been well recognized as an important neuroprotective agent in a variety of neurological disorders in the central nervous system (CNS), such as spinal cord injury (SCI), traumatic brain injury (TBI), Alzheimer's disease, and multiple sclerosis. The exact molecular mechanisms of EST-mediated neuroprotection in the CNS remain unclear due to heterogeneity of cell populations that express EST receptors (ERs) in the CNS as well as in the innate and adaptive immune system. Recent investigations suggest that EST protects the CNS from injury by suppressing pro-inflammatory pathways, oxidative stress, and cell death, while promoting neurogenesis, angiogenesis, and neurotrophic support. In this review, we have described the currently known molecular mechanisms of EST-mediated neuroprotection and neuroregeneration in SCI and TBI. At the same time, we have emphasized on the recent in vitro and in vivo findings from our and other laboratories, implying potential clinical benefits of EST in the treatment of SCI and TBI.

RIP1 and RIP3 complex regulates radiation-induced programmed necrosis in glioblastoma.

Radiation-induced necrosis (RN) is a relatively common side effect of radiation therapy for glioblastoma. However, the molecular mechanisms involved and the ways RN mechanisms differ from regulated cell death (apoptosis) are not well understood. Here, we compare the molecular mechanism of cell death (apoptosis or necrosis) of C6 glioma cells in both in vitro and in vivo (C6 othotopically allograft) models in response to low and high doses of X-ray radiation. Lower radiation doses were used to induce apoptosis, while high-dose levels were chosen to induce radiation necrosis. Our results demonstrate that active caspase-8 in this complex I induces apoptosis in response to low-dose radiation and inhibits necrosis by cleaving RIP1 and RI. When activation of caspase-8 was reduced at high doses of X-ray radiation, the RIP1/RIP3 necrosome complex II is formed. These complexes induce necrosis through the caspase-3-independent pathway mediated by calpain, cathepsin B/D, and apoptosis-inducing factor (AIF). AIF has a dual role in apoptosis and necrosis. At high doses, AIF promotes chromatinolysis and necrosis by interacting with histone H2AX. In addition, NF-κB, STAT-3, and HIF-1 play a crucial role in radiation-induced inflammatory responses embedded in a complex inflammatory network. Analysis of inflammatory markers in matched plasma and cerebrospinal fluid (CSF) isolated from in vivo specimens demonstrated the upregulation of chemokines and cytokines during the necrosis phase. Using RIP1/RIP3 kinase specific inhibitors (Nec-1, GSK'872), we also establish that the RIP1-RIP3 complex regulates programmed necrosis after either high-dose radiation or TNF-α-induced necrosis requires RIP1 and RIP3 kinases. Overall, our data shed new light on the relationship between RIP1/RIP3-mediated programmed necrosis and AIF-mediated caspase-independent programmed necrosis in glioblastoma.

Administration of low dose estrogen attenuates gliosis and protects neurons in acute spinal cord injury in rats.

Spinal cord injury (SCI) is a debilitating condition with neurological deficits and loss of motor function that, depending on the severity, may lead to paralysis. The only treatment currently available is methylprednisolone, which is widely used and renders limited efficacy in SCI. Therefore, other therapeutic agents must be developed. The neuroprotective efficacy of estrogen in SCI was studied with a pre-clinical and pro-translational perspective. Acute SCI was induced in rats that were treated with low doses of estrogen (1, 5, 10, or 100 µg/kg) and compared with vehicle-treated injured rats or laminectomy control (sham) rats at 48 h post-SCI. Changes in gliosis and other pro-inflammatory responses, expression and activity of proteolytic enzymes (e.g., calpain, caspase-3), apoptosis of neurons in SCI, and cell death were monitored via Western blotting and immunohistochemistry. Negligible pro-inflammatory responses or proteolytic events and very low levels of neuronal death were found in sham rats. In contrast, vehicle-treated SCI rats showed profound pro-inflammatory responses with reactive gliosis, elevated expression and activity of calpain and caspase-3, elevated Bax:Bcl-2 ratio, and high levels of neuronal death in lesion and caudal regions of the injured spinal cord. Estrogen treatment at each dose reduced pro-inflammatory and proteolytic activities and protected neurons in the caudal penumbra in acute SCI. Estrogen treatment at 10 µg was found to be as effective as 100 µg in ameliorating the above parameters in injured animals. Results from this investigation indicated that estrogen at a low dose could be a promising therapeutic agent for treating acute SCI. Experimental studies with low dose estrogen therapy in acute spinal cord injury (SCI) demonstrated the potential for multi-active beneficial outcomes. Estrogen has been found to ameliorate several degenerative pathways following SCI. Thus, such early protective effects may even lead to functional recovery in long term injury. Studies are underway in chronic SCI in a follow up manuscript.