Four adult nonhuman primates (Macaca mulatta) underwent bilateral thalamic gray matter or frontal white matter infusions. All procedures were performed in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee of the National Institute of Neurological Disorders and Stroke.
Preparation of M13 Bacteriophage and Gd-DTPA Infusate
The M13 bacteriophage (NeuroPhage Pharmaceuticals, Inc.) was diluted with PBS to a final concentration of 1013
viral particles/ml. Clinical-grade Gd-DTPA (Bayer Pharmaceuticals) was also diluted with bacteriophage in fusate to a final concentration of 1 mM. The dilutions were prepared 1 hour before infusion.
Surgical Placement of Infusion Pedestal
We used MRI-compatible infusion pedestals as previously described by Rosenbluth et al.22
Approximately 1–14 days before infusion, general endotracheal anesthesia (0.5%–3% isoflurane) was induced in each primate, and each was intubated for the stereotactic placement of the infusion pedestal. Using sterile surgical technique, a midline incision was made, exposing the underlying muscle and calvaria. A 12.5-mm bur hole was made directly over the target site, exposing the underlying dura mater. The infusion pedestal was placed at an appropriate angle (as determined by preoperative MRI) and secured using nylon screws and fixative cement. Covering caps were placed over the pedestals.
Convective Coinfusion of M13 Bacteriophage and Gd-DTPA
Coinfusion of M13 bacteriophage and Gd-DTPA was performed as previously described.14
Briefly, bilateral thalamic gray matter (4 infusions) or frontal white matter (3 infusions) was coinfused (Vi 65–70 μl and 25–30 μl M13 bacteriophage and Gd-DTPA, respectively) (). Infusion times ranged from approximately 70 minutes in white matter to 155 minutes in gray matter. During infusion, animals were anesthetized and placed into a stereotactic frame (Crist Instrument Co., Inc.). A fused silica step cannula,22
which was made by inserting a 28-gauge (0.3-mm inner diameter) into a 22-gauge (0.6-mm outer diameter) infusion cannula, was stereotactically placed through the previously implanted pedestal into the target structure. Infusions were initially started at 0.1 μl/minute and then increased to 0.5 μl/minute for the duration of the infusion until the desired infusion volume was attained. After recovery from anesthesia, the animals were monitored for neurological or behavioral changes resulting from the infusions into the targeted areas. Three days after infusion the animals were killed. The brain was perfused and removed for sectioning and histological studies.
Distribution of M13 bacteriophage by CED in 4 nonhuman primates*
Tracking of Infusion Using MRI
Immediately before infusion, MRI coordinates (T1-weighted, coronal images) were obtained to confirm accurate cannula placement at the target site of interest (thalamic gray matter or frontal white matter). During the infusions, coronal T1-weighted MRI studies (slice thickness 1 mm without spacing) were obtained on a 3-T machine constantly at 8-minute intervals until infusion completion.
Analysis of MRI Findings for Gd-DTPA Distribution
As previously described,14
the Vd of Gd-DTPA on the MRI studies was analyzed using a Sun Microsystems, Inc. workstation running MEDx 3.4 software (Sensor Systems, Inc.). A 10% threshold segmentation analysis was performed to calculate the Vd of imaged Gd-DTPA. This Vd was compared with the Vi to obtain the Vd:Vi ratio.
Analysis of M13 Bacteriophage Distribution and Perfused Tissue
Three days after infusion, primates were killed with administration of intravenous sodium pentobarbital (90 mg/kg). Transcardiac perfusion was performed with 0.5 L of 10% heparinized saline and 1 L of 4% paraformaldehyde. Brains were removed after perfusion and fixed in 4% paraformaldehyde. Brains were then washed with PBS, placed in a 30% sucrose solution in 0.01 M PBS for cryopreservation, and then sectioned through the infusion site at 40 μm on a freezing sliding microtome.
Immunostaining for M13 bacteriophage was performed on every sixth serial section (40-μm thickness). Each section was washed 3 times in PBS and blocked in 1% hydrogen peroxide. Sections were washed again in PBS and blocked using background sniper (Biocare Medical). The sections were incubated with primary monoclonal mouse anti-M13 antibody (GE Healthcare, Inc.) overnight. After washing, the sections were incubated with secondary MACH 2 mouse HRP-polymer antibody (Biocare Medical) for 1 hour. The reaction product was visualized using diaminobenzidine and hydrogen peroxide, and tissue was mounted on glass slides, dehydrated, and coverslipped with mounting media.
Fixed and coverslipped slides were digitized on low-power microscopy using AxioVision software (version 4.8; Carl Zeiss, Inc.), and the distribution of M13 bacteriophage was determined using ImageJ software (NIH ImageJ, version 1.44). The Cavalieri method was used to calculate the Vd from serial sections immunostained for M13 bacteriophage.
We performed H & E and Nissl staining on adjacent representative sections extending from the cannula and infusion sites for a qualitative assessment of changes in tissue architecture and toxicity.
Excel (Microsoft Corp.) and Prism (Graphpad Software) software programs were used for statistical analysis and graphical representation of the results. A Student t-test was performed, with a p value set at 0.05.