scholarly journals Distribution and stability of antisense phosphorothioate oligonucleotides in rodent brain following direct intraparenchymal controlled-rate infusion

1997 ◽  
Vol 3 (5) ◽  
pp. E6 ◽  
Author(s):  
William C. Broaddus ◽  
Sujit S. Prabhu ◽  
George T. Gillies ◽  
Jeffrey Neal ◽  
William S. Conrad ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Structural Changes ◽  
Tissue Concentration ◽  
Volume Of Distribution ◽  
Brain Parenchyma ◽  
Average Concentration ◽  
High Flow ◽  
Fischer 344 ◽  
High Concentrations ◽  
The Brain

High-flow microinfusion is a novel technique for delivery of compounds directly into the brain parenchyma, bypassing the blood-brain barrier. The feasibility of this technique has been demonstrated with low-molecular-weight compounds, macromolecular dyes, and proteins. Delivery of antisense oligonucleotides into the brain parenchyma represents an additional potential application of this technique not previously described. In this report, the authors examined the distribution and disposition of phosphorothioate oligodeoxynucleotide (PS-ODN) infused for this reason. An 18-mer 35S-PS-ODN (molecular weight approximately 6000) was infused over 1 hour into the caudate putamen of Fischer 344 rats. At 1, 6, 12, 24, and 48 hours after beginning the infusion, the brains were extracted and analyzed using quantitative autoradiographic techniques. Cerebrospinal fluid (CSF) was also aspirated from the cisterna magna and analyzed for radioactivity and stability of the 35S-PS-ODN. At 1 hour, the infused ODN was uniformly distributed in brain tissue, with a maximum average concentration of 4806.5 ± 210.5 nCi/g. This represents a tissue concentration of 19.2 ± 0.84 μM. Extensive spread into surrounding parenchyma was observed over the ensuing 47 hours. The 35S-PS-ODN radioactivity peaked in the CSF at the end of the 1-hour infusion, containing 10% (50 ± 20 nCi) of the infused radioactivity. Activity then decayed exponentially over 11 hours, stabilizing at a lower CSF content of 0.2% (1 ± 0.1 nCi). The volume of distribution (Vd) was 105 ± 7.9 mm3 at 1 hour, representing a ratio of Vd/Vi (volume of infusion) of 5.2. The Vd increased to 443.4 ± 62.3 mm3 at the end of 48 hours, whereas the average minimum tissue concentration decreased from 15.2 to 3.2 μM. Undegraded 18-mer was seen throughout the 48-hour period using 20% polyacrylamide/7M urea gel electrophoresis. The animals tolerated the infusion without evidence of toxicity, and minimal structural changes in tissue were observed on histological examination. Thus, PS-ODN can be safely delivered in high concentrations to wide areas of the rat brain by using high-flow microinfusion, and the concentrations remain stable even after 48 hours in situ.

Distribution and stability of antisense phosphorothioate oligonucleotides in rodent brain following direct intraparenchymal controlled-rate infusion

1998 ◽  
Vol 88 (4) ◽  
pp. 734-742 ◽  
Author(s):  
William C. Broaddus ◽  
Sujit S. Prabhu ◽  
George T. Gillies ◽  
Jeffrey Neal ◽  
William S. Conrad ◽  
...  
Keyword(s):  
Structural Changes ◽  
Tissue Concentration ◽  
Volume Of Distribution ◽  
Brain Parenchyma ◽  
Average Concentration ◽  
High Flow ◽  
Content Type ◽  
Fischer 344 ◽  
High Concentrations ◽  
Fine Print

Object. High-flow microinfusion is a novel technique for delivery of compounds directly into brain parenchyma, bypassing the blood-brain barrier. The feasibility of this technique has been demonstrated with low-molecular-weight compounds, macromolecular dyes, and proteins. Delivery of antisense oligonucleotides into brain parenchyma represents an additional potential application of this technique not previously described. In this report the authors sought to examine the distribution and disposition of phosphorothioate oligodeoxynucleotide (PS-ODN) for this reason. Methods. An 18-mer 35S-PS-ODN (M r approximately 6000) was infused over 1 hour into the caudate putamen of Fischer 344 rats. At 1, 6, 12, 24, and 48 hours after beginning the infusion, the brains were extracted and analyzed using quantitative autoradiographic techniques. Cerebrospinal fluid (CSF) was also aspirated from the cisterna magna and was analyzed to determine the radioactivity and stability of the 35S-PS-ODN. At 1 hour, the infused ODN was uniformly distributed in brain tissue, with a maximum average concentration of 4806.5 ± 210.5 nCi/g. This represents a tissue concentration of 19.2 ± 0.84 µM. Extensive spread into surrounding parenchyma was observed over the ensuing 47 hours. The 35S-PS-ODN radioactivity peaked in the CSF at the end of the 1-hour infusion, containing 1% (50 ± 20 nCi) of the infused radioactivity. Activity then decayed exponentially over 11 hours, but stabilized at a lower CSF content of 0.2% (1 ± 0.1 nCi) thereafter. The volume of distribution was 105 ± 7.9 mm3 at 1 hour, representing a volume of distribution/volume of infusion ratio of 5.2. The volume of distribution increased to 443 ± 62.3 mm3 at the end of 48 hours, whereas the average minimum tissue concentration decreased from 15.2 µM to 3.2 µM. Undegraded 18-mer was observed throughout the 48-hour period by means of 20% polyacrylamide/7 M urea gel electrophoresis. The animals tolerated the infusion without evidence of toxicity and minimal structural changes in tissue were observed on histological investigation. Conclusions. The authors found that PS-ODNs can be safely delivered in high concentrations to wide areas of rat brain by using high-flow microinfusion and are stable even after 48 hours in situ.

Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

2020 ◽  
Author(s):  
Katie Mae Wilson ◽  
Aurora Burkus-Matesevac ◽  
Samuel Maddox ◽  
Christopher Chouinard
Keyword(s):  
Structural Changes ◽  
Noncovalent Complex ◽  
Unfolded Protein ◽  
Protein Size ◽  
High Concentrations ◽  
The Unfolded Protein Response ◽  
Critical Binding ◽  
Protein Response ◽  
The Brain ◽  
Lateral Sclerosis

β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.

Native Ubiquitin Structural Changes Resulting from Complexation with β-methylamino-L-alanine (BMAA)

2020 ◽  
Author(s):  
Katie Mae Wilson ◽  
Aurora Burkus-Matesevac ◽  
Samuel Maddox ◽  
Christopher Chouinard
Keyword(s):  
Structural Changes ◽  
Noncovalent Complex ◽  
Unfolded Protein ◽  
Protein Size ◽  
High Concentrations ◽  
The Unfolded Protein Response ◽  
Critical Binding ◽  
Protein Response ◽  
The Brain ◽  
Lateral Sclerosis

β-methylamino-L-alanine (BMAA) has been linked to the development of neurodegenerative (ND) symptoms following chronic environmental exposure through water and dietary sources. The brains of those affected by this condition, often referred to as amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), have exhibited the presence of plaques and neurofibrillary tangles (NFTs) from protein aggregation. Although numerous studies have sought to better understand the correlation between BMAA exposure and onset of ND symptoms, no definitive link has been identified. One prevailing hypothesis is that BMAA acts a small molecule ligand, complexing with critical proteins in the brain and reducing their function. The objective of this research was to investigate the effects of BMAA exposure on the native structure of ubiquitin. We hypothesized that formation of a Ubiquitin+BMAA noncovalent complex would alter the protein’s structure and folding and ultimately affect the ubiquitinproteasome system (UPS) and the unfolded protein response (UPR). Ion mobility-mass spectrometry revealed that at sufficiently high concentrations BMAA did in fact form a noncovalent complex with ubiquitin, however similar complexes were identified for a range of additional amino acids. Collision induced unfolding (CIU) was used to interrogate the unfolding dynamics of native ubiquitin and these Ubq-amino acid complexes and it was determined that complexation with BMAA led to a significant alteration in native protein size and conformation, and this complex required considerably more energy to unfold. This indicates that the complex remains more stable under native conditions and this may indicate that BMAA has attached to a critical binding location.

scholarly journals PDTM-30. UNDERSTANDING THE EFFECTS OF MOLECULAR SIZE ON VOLUME OF DISTRIBUTION IN CONVECTION-ENHANCED DELIVERY

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi193-vi194
Author(s):  
Erica Power ◽  
Julian Rechberger ◽  
Liang Zhang ◽  
David Daniels
Keyword(s):  
Molecular Weight ◽  
Single Injection ◽  
Fluorescent Microscopy ◽  
Molecular Size ◽  
Volume Of Distribution ◽  
Sprague Dawley ◽  
Convection Enhanced Delivery ◽  
Microscopy Imaging ◽  
Delivery Technique ◽  
The Brain

Abstract BACKGROUND Diffuse midline gliomas harboring the H3K27M mutation, previously known as diffuse intrinsic pontine gliomas (DIPG), are rare and aggressive pediatric brain tumors. Over 100 clinical trials with different chemotherapeutics have failed to show any therapeutic benefit. One reason for failure is likely due to poor delivery of these agents to the brainstem. Convection-enhanced delivery (CED) is an emerging delivery technique used to directly inject the agent of interest into the brainstem under pressure. While there is evidence that this may be an effective delivery method, little work has been done to understand the optimal physical properties of these drugs. We sought characterize volume of distribution in the brain based on molecular size of the agent delivered via CED. METHODS Sprague- Dawley rats underwent a single injection of FITC-dextran (3,000 Da, 10,000 Da, 20,000 Da, 70,000 Da, 150,000 Da) via CED into the pons. Post-injection, animals were sacrificed and their brains harvested. Fluorescent microscopy imaging was used to calculate the volume of distribution of the FITC-dextran throughout the brain. RESULTS The volume of distribution (Vd) decreased exponentially according to a two-phase delay (r2= 0.94) as the molecular size of the FITC-dextran increased. The highest mean Vd (107.87mm3) was at a molecular weight of 3,000 Da, and lowest mean Vd (26.48 mm3) was at a molecular weight of 150,000 Da. ANOVA analysis was statistically significant (p= 0.0017). CONCLUSIONS As the molecular size of the FITC-dextran increased, the volume of distribution within the brain following a single injection via CED into the pons decreased. A better understanding of how drugs distribute by convection will allow us to optimize treatment regimens for DIPG tumors.

scholarly journals Aβ delays fibrin clot lysis by altering fibrin structure and attenuating plasminogen binding to fibrin

Blood ◽  
2012 ◽  
Vol 119 (14) ◽  
pp. 3342-3351 ◽  
Author(s):  
Daria Zamolodchikov ◽  
Sidney Strickland
Keyword(s):  
Structural Changes ◽  
Fibrin Clot ◽  
Brain Parenchyma ◽  
Amyloid Peptide ◽  
Clot Lysis ◽  
Fibrin Network ◽  
Β Amyloid Peptide ◽  
Clot Structure ◽  
The Brain ◽  
Fibrin Clots

Abstract Alzheimer disease is characterized by the presence of increased levels of the β-amyloid peptide (Aβ) in the brain parenchyma and cerebral blood vessels. This accumulated Aβ can bind to fibrin(ogen) and render fibrin clots more resistant to degradation. Here, we demonstrate that Aβ42 specifically binds to fibrin and induces a tighter fibrin network characterized by thinner fibers and increased resistance to lysis. However, Aβ42-induced structural changes cannot be the sole mechanism of delayed lysis because Aβ overlaid on normal preformed clots also binds to fibrin and delays lysis without altering clot structure. In this regard, we show that Aβ interferes with the binding of plasminogen to fibrin, which could impair plasmin generation and fibrin degradation. Indeed, plasmin generation by tissue plasminogen activator (tPA), but not streptokinase, is slowed in fibrin clots containing Aβ42, and clot lysis by plasmin, but not trypsin, is delayed. Notably, plasmin and tPA activities, as well as tPA-dependent generation of plasmin in solution, are not decreased in the presence of Aβ42. Our results indicate the existence of 2 mechanisms of Aβ42 involvement in delayed fibrinolysis: (1) through the induction of a tighter fibrin network composed of thinner fibers, and (2) through inhibition of plasmin(ogen)–fibrin binding.

scholarly journals ‘Tanned’ gelatin spheres and granules for exclusion chromatography

1968 ◽  
Vol 108 (4) ◽  
pp. 641-646 ◽  
Author(s):  
A. Polson ◽  
W. Katz
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
High Molecular Weight ◽  
Weight Fraction ◽  
High Flow ◽  
High Molecular Weight Fraction ◽  
Flow Rates ◽  
Protein Molecules ◽  
Exclusion Chromatography

1. The preparation of tanned gelatin spheres and granules from high-molecular-weight gelatin is described. This material is comparatively hard, giving high flow rates, is insoluble in water at temperatures between 0° and 100° and is resistant to digestion by trypsin and chymotrypsin. The high-molecular-weight fraction of gelatin was prepared by precipitation with polyethylene glycol, and the spheres and granules prepared from this fraction were hardened and insolubilized by tanning with either formalin or chromium salts or both. 2. The spheres and granules were used successfully for the separation of protein molecules and other protein-aceous materials ranging in molecular weight from 200 to greater than 6000000. This gel exclusion material has several properties superior to those of other products used for similar purposes. Further, it was noticed that the porosity of the spheres differed considerably from that of the granules.

scholarly journals Macrophages and microglia: the cerberus of glioblastoma

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Alice Buonfiglioli ◽  
Dolores Hambardzumyan
Keyword(s):  
Tumor Cells ◽  
Innate Immune System ◽  
Tumor Heterogeneity ◽  
Expression Profiles ◽  
Brain Parenchyma ◽  
Innate Immune ◽  
Malignant Growth ◽  
Neoplastic Cells ◽  
Functional Roles ◽  
The Brain

AbstractGlioblastoma (GBM) is the most aggressive and deadliest of the primary brain tumors, characterized by malignant growth, invasion into the brain parenchyma, and resistance to therapy. GBM is a heterogeneous disease characterized by high degrees of both inter- and intra-tumor heterogeneity. Another layer of complexity arises from the unique brain microenvironment in which GBM develops and grows. The GBM microenvironment consists of neoplastic and non-neoplastic cells. The most abundant non-neoplastic cells are those of the innate immune system, called tumor-associated macrophages (TAMs). TAMs constitute up to 40% of the tumor mass and consist of both brain-resident microglia and bone marrow-derived myeloid cells from the periphery. Although genetically stable, TAMs can change their expression profiles based upon the signals that they receive from tumor cells; therefore, heterogeneity in GBM creates heterogeneity in TAMs. By interacting with tumor cells and with the other non-neoplastic cells in the tumor microenvironment, TAMs promote tumor progression. Here, we review the origin, heterogeneity, and functional roles of TAMs. In addition, we discuss the prospects of therapeutically targeting TAMs alone or in combination with standard or newly-emerging GBM targeting therapies.

scholarly journals Practical application of synthetic head models in real ballistic cases

2021 ◽  
Author(s):  
F. Riva ◽  
T. Fracasso ◽  
A. Guerra ◽  
P. Genet
Keyword(s):  
Soft Tissues ◽  
Spherical Model ◽  
Brain Parenchyma ◽  
Human Bone ◽  
Shape Model ◽  
The Difference ◽  
Open Shape ◽  
The Brain ◽  
Synthetic Models ◽  
Representative Material

AbstractIn shooting crimes, ballistics tests are often recommended in order to reproduce the wound characteristics of the involved persons. For this purpose, several “simulants” can be used. However, despite the efforts in the research of “surrogates” in the field of forensic ballistic, the development of synthetic models needs still to be improved through a validation process based on specific real caseworks. This study has been triggered by the findings observed during the autopsy performed on two victims killed in the same shooting incident, with similar wounding characteristics; namely two retained head shots with ricochet against the interior wall of the skull; both projectiles have been recovered during the autopsies after migration in the brain parenchyma. The thickness of the different tissues and structures along the bullets trajectories as well as the incident angles between the bullets paths and the skull walls have been measured and reproduced during the assemblage of the synthetic head models. Two different types of models (“open shape” and “spherical”) have been assembled using leather, polyurethane and gelatine to simulate respectively skin, bone and soft tissues. Six shots have been performed in total. The results of the models have been compared to the findings of post-mortem computed tomography (PMCT) and the autopsy findings.Out of the six shots, two perforated the models and four were retained. When the projectile was retained, the use of both models allowed reproducing the wounds characteristics observed on both victims in terms of penetration and ricochet behaviour. However, the projectiles recovered from the models showed less deformation than the bullets collected during the autopsies. The “open shape” model allowed a better controlling on the shooting parameters than the “spherical” model. Finally, the difference in bullet deformation could be caused by the choice of the bone simulant, which might under-represent either the strength or the density of the human bone. In our opinion, it would be worth to develop a new, more representative material for ballistic which simulates the human bone.

scholarly journals Primary Cranial Vault Lymphoma Extending between Subcutaneous Tissue and Brain Parenchyma without Skull Destruction after Mild Head Trauma: A Case Report and Literature Review

2021 ◽  
pp. 1118-1123
Author(s):  
Kengo Setta ◽  
Takaaki Beppu ◽  
Yuichi Sato ◽  
Hiroaki Saura ◽  
Junichi Nomura ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Head Trauma ◽  
Dura Mater ◽  
Subcutaneous Tissue ◽  
Bone Destruction ◽  
B Cell Lymphoma ◽  
Brain Parenchyma ◽  
Skin Tumor ◽  
Cranial Vault ◽  
The Brain

Malignant lymphoma of the head rarely arises outside of the brain parenchyma as primary cranial vault lymphoma (PCVL). A case of PCVL that invaded from subcutaneous tissue into the brain, passing through the skull, and occurred after mild head trauma is reported along with a review of the literature. The patient was a 75-year-old man with decreased activity. One month before his visit to our hospital, he bruised the left frontal area of his head. Magnetic resonance imaging showed homogeneously enhanced tumors with contrast media in the subcutaneous tissue corresponding to the head impact area and the cerebral parenchyma, but no obvious abnormal findings in the skull. A biopsy with craniotomy was performed under general anesthesia. The pathological diagnosis was diffuse large B-cell lymphoma. On histological examination, tumor cells grew aggressively under the skin. Tumor cells invaded along the emissary vein into the external table without remarkable bone destruction and extended across the skull through the Haversian canals in the diploe. Tumor cells were found only at the perivascular areas in the dura mater and extended into the brain parenchyma. Considering the history of head trauma and the neuroimaging and histological findings, the PCVL in the present case arose primarily under the skin, passed though the skull and dura mater, and invaded along vessels and reached the brain.

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