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Zhi-Jian Chen, George T. Gillies, William C. Broaddus, Sujit S. Prabhu, Helen Fillmore, Ryan M. Mitchell, Frank D. Corwin and Panos P. Fatouros

Object. The goal of this study was to validate a simple, inexpensive, and robust model system to be used as an in vitro surrogate for in vivo brain tissues in preclinical and exploratory studies of infusion-based intraparenchymal drug and cell delivery.

Methods. Agarose gels of varying concentrations and porcine brain were tested to determine the infusion characteristics of several different catheters at flow rates of 0.5 and 1 µl per minute by using bromophenol blue (BPB) dye (molecular weight [MW] ∼690) and gadodiamide (MW ∼573). Magnetic resonance (MR) imaging and videomicroscopy were used to measure the distribution of these infusates, with a simultaneous measurement of infusion pressures. In addition, the forces of catheter penetration and movement through gel and brain were measured.

Agarose gel at a 0.6% concentration closely resembles in vivo brain with respect to several critical physical characteristics. The ratio of distribution volume to infusion volume of agarose was 10 compared with 7.1 for brain. The infusion pressure of the gel demonstrated profiles similar in configuration and magnitude to those of the brain (plateau pressures 10–20 mm Hg). Gadodiamide infusion in agarose closely resembled that in the brain, as documented using T1-weighted MR imaging. Gadodiamide distribution in agarose gel was virtually identical to that of BPB dye, as documented by MR imaging and videomicroscopy. The force profile for insertion of a silastic catheter into agarose gel was similar in magnitude and configuration to the force profile for insertion into the brain. Careful insertion of the cannula using a stereotactic guide is critical to minimize irregularity and backflow of infusate distribution.

Conclusions. Agarose gel (0.6%) is a useful surrogate for in vivo brain in exploratory studies of convection-enhanced delivery.

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William C. Broaddus, Sujit S. Prabhu, George T. Gillies, Jeffrey Neal, William S. Conrad, Zhi-Jian Chen, Helen Fillmore and Harold F. Young

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.

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William C. Broaddus, Yue Liu, Laura L. Steele, George T. Gillies, Peck-Sun Lin, William G. Loudon, Kristoffer Valerie, Rupert K. Schmidt-Ullrich and Helen L. Fillmore

Object. The goal of this study was to determine whether adenoviral vector—mediated expression of human wildtype p53 can enhance the radiosensitivity of malignant glioma cells that express native wild-type p53.

The p53 gene is thought to function abnormally in the majority of malignant gliomas, although it has been demonstrated to be mutated in only approximately 30%. This has led to studies in which adenoviral transduction with wild-type human p53 has been investigated in an attempt to slow tumor cell growth. Recent studies suggest that reconstitution of wild-type p53 can render cells more susceptible to radiation-mediated death, primarily by p53-mediated apoptosis.

Methods. Rat RT2 glioma cells were analyzed for native p53 status by reverse transcriptase—polymerase chain reaction and sequence analysis and for p53 expression by Western blot analysis. Clonogenic survival and the terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling assay were used to characterize RT2 cell radiosensitivity and apoptosis, respectively, with and without prior transduction with p53-containing and control adenoviral vectors. Animal survival length was monitored after intracerebral implantation with transduced and nontransduced RT2 cells, with and without cranial radiation.

The RT2 cells were demonstrated to express native rat wild-type p53 and to markedly overexpress human p53 following adenoviral p53 transduction. The combination of p53 transduction followed by radiation resulted in marked decreases in RT2 cell survival and increases in apoptosis at radiation doses from 2 to 6 Gy. Animals receiving cranial radiation after intracerebral implantation with RT2 cells previously transduced with p53 survived significantly longer than control animals (p < 0.01).

Conclusions. The ability to enhance the radiosensitivity of malignant glioma cells that express wild-type p53 by using adenoviral transduction to induce overexpression of p53 offers hope for this approach as a therapeutic strategy, not only in human gliomas that express mutant p53, but also in those that express wild-type p53.

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Gregory A. Helm, Jonas M. Sheehan, Jason P. Sheehan, John A. Jane Jr., Charles G. diPierro, Nathan E. Simmons, George T. Gillies, David F. Kallmes and Thomas M. Sweeney

✓ Autologous bone grafts are currently considered “gold standard” material for achieving long-term spinal arthrodesis. The present study was performed to determine whether demineralized bone matrix (DBM), type I collagen gels, or bone morphogenetic protein-2 (BMP-2) can improve autologous bone spinal fusions. Using a unilateral decompression—contralateral fusion technique in dogs, each of these materials was added to an autologous bone graft. Volumetric analysis, histological analysis, and biomechanical testing were performed to assess the effectiveness of each material. The DBM had an inhibitory effect on solid bone fusion of the spine, whereas the type I collagen gels improved the bony interface between the graft and the host spine. The BMP-2 strongly enhanced the amount of bone deposition at the fusion site and increased the number of intervertebral levels that were solidly fused. This study strongly supports the use of BMP-2 as an additive to autologous bone grafts in spine stabilization.

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William C. Broaddus, Sujit S. Prabhu, George T. Gillies, Jeffrey Neal, William S. Conrad, Zhi-Jian Chen, Helen Fillmore and Harold F. Young

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.