In vivo spatiotemporal dynamics of NG2 glia activity caused by neural electrode implantation.

Surgical probe implantation

Two-photon experiments were conducted using four-shank 16-channel Michigan style silicon probes (3 mm long, 15 μm thick, 55 μm wide, 125 μm center-to-center shank spacing) mounted on non-functional tabs (NeuroNexus Technologies, Ann Arbor, MI). Electrodes were implanted into the cerebral cortex of transgenic mice (male, 22-30g, Jackson Laboratories; Bar Harbor, ME) expressing either green fluorescent protein (GFP) in NG2-glia ( Cspg4-EGFP , n=5) or GFP in microglia cells ( CX3cr1-EGFP , n=5). Prior to surgery animals were administered an intraperitoneal (IP) injection of 75 mg/kg ketamine and 7 mg/kg xylazine cocktail and placed in a stereotaxic frame. After removing the skin and connective tissue from the skull, a thin layer of Vetbond (3M) was applied to dry the bone and improve adhesion between the bone and headcap. Two bone screws were inserted into bone screw holes made using a high-speed dental drill over both motor cortices and secured with dental cement. A 4 mm by 6 mm craniotomy was performed over the left visual cortex centered at a point 1.5 mm rostral to lambda and 1 mm lateral from the midline. To prevent thermal damage from drilling, the skull was periodically bathed in saline. Probes were inserted through intact dura and pia in the rostral direction into the cortex at a 30° angle and parallel to the midline at 400 μm/s for a total distance of 600 μm (oil hydraulic Microdrive; MO-82, Narishige, Japan) and a final resting depth of 250-300 μm (layer II-III) beneath the surface of the brain. Effort was taken to avoid blood vessel penetration during insertion. To create a chronic imaging window, Kwik-Sil was used as a sealant inside the craniotomy before placing a glass coverslip and securing with dental cement[ 33 ]. A 2 mm tall well was formed around the imaging window to hold water for a water-immersive objective lens. Sulforhodamine 101 (SR101) was injected IP to visualize vascularization (red; 0.02-0.04 cc; 1 mg of drug per ml of sterile saline; taken from [ 34 ]). Updates of SR101 were administered approximately every hour to maintain vascular labeling. All procedures and experimental protocols were approved by the University of Pittsburgh, Division of Laboratory Animal Resources, and Institutional Animal Care and Use Committee in accordance with the standards for humane animal care as set by the Animal Welfare Act and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Two-Photon Imaging

In vivo imaging was performed using a two-photon laser scanning microscope as previously published [ 34 ]. The microscope consisted of a scan head (Bruker, Madison, WI), an OPO laser (Insight DS+; Spectra-Physics, Menlo Park, CA) tuned to a wavelength of 920 nm, non-descanned photomultiplier tubes (Hamamatsu Photonics KK, Hamamatsu, Shizuoka, Japan), and a 16X, 0.8 numerical aperture water immersion objective lens (Nikon Instruments, Melville, NY). During imaging, mice were anesthetized with isoflurane (1.0-1.5%, mixed with 0.9 L/min O 2 ) and, prior to every Z-stack, injected with Sulforhodamine 101 (SR101) for visualization of blood vessels. If an animal experienced pial surface bleeding severe enough to impact imaging quality, the animal was removed from the study. An imaging window of 407.5 × 407.5 μm (1024 × 1024 pixels) was used to visualize electrode shanks and adjacent tissue for quantification. Regions of interest were chosen by judging the two outermost shanks for the least amount of vasculature within the adjacent tissue to maximize image clarity. Z-stack images every 2 μm along the full depth of the implant were acquired at 2, 4, 6, 8, 10, 12, 24, 48 and 72 hours post-insertion with a scan rate of ~5 s/image.

Data Analysis

Image z-stacks were processed using ImageJ (National Institute of Health). In order to quantify differences in cell processes extension, soma migration, and morphology, z-stacks of consecutive time points were aligned with each other with respect to the probe. To do this, the offset position of one z-stack relative to another z-stack was determined using a “TurboReg” plugin for ImageJ. Next, the translate feature was used to align both z-stacks with each other. Cell processes and soma migration were quantified up until the point of destination at the surface of the implant. Both processes and cell body movements moving toward or away from the probe were tracked by determining XY coordinates and using the ‘Measure’ feature in ImageJ. Direction of cell processes extension and soma movement was noted by drawing a line through the center of the cell parallel to the surface of the probe. Processes extension was recorded as the distance between processes for consecutive time points while migration of cell bodies was recorded as the distance between soma for consecutive time points. Surface coverage of NG2 glia and microglial processes along the face of the probe were measured as the percent of fluorescent signal measured over the total probe area. Each z-stack was rotated using ImageJ’s built-in plugin “Interactive Stack Rotation” to reslice the total volume of tissue normal to the probe surface. A binary mask of the sum slice projection of a small volume of tissue directly above the probe surface was created by using a built-in ImageJ thresholding method utilizing an isodata algorithm [ 35 ]. The outline of the probe was identified and the number of nonzero pixels were counted and taken as a fraction of the total area measured. The outline defined around the probe was verified to ensure correct dimensions around the probe perimeter. Since the NG2 protein is also expressed in pericytes and macrophages as well as NG2 glia [ 36 ], examination of each tracked cell for a multi-processed morphology, a characteristic unique to NG2+ oligodendrocyte precursor cells, was confirmed before analysis. Cell morphology was characterized using morphological metrics developed previously [ 34 ]. Cells were classified as either ramified, or non-activated (1), with processes extending equally in every direction, or in a transitional (activated) stage (0), with processes orientated preferentially on one side of the soma. This morphological state was determined using the same hemispheric divide previously detailed for cell processes and soma velocity. Cells were binned based on the distance between the center of soma and the probe surface. A logistic regression was used to fit the data to show the Bernoulli Probability Distribution of cells in a ramified or transitional state (0 or 1) as a function of distance from the surface of the probe as previously published [ 34 ]. Two other metrics, the transitional index (T-index) and directionality index (D-index), were used to quantify and compare cell morphology. The T-index was calculated by measuring the length of the leading process ( n ), which is the longest process extending toward the probe, and the length of the longest lagging process ( f ), which is the process extended away from the probe at each time point. The D-index was calculated in a similar manner by counting the number of processes extending toward the probe ( n ) and the number of processes orientated away from the probe ( f ) using the hemispheric divide to distinguish between toward and away processes. The following formula was used to calculate index values for each time point: (1) Index = ( f − n ) ( f + n ) + 1. For both T-index and D-index, a measured index of 1 indicates a ramified, or non-activated, morphology while an index of 0 indicates a fully transitional, or activated, morphology with processes extending toward the surface of the probe, similar to the ramification method detailed previously [ 34 ]. After binning, cell morphology was reported as a distribution of index values as a function of distance from the probe surface. A dual sigmoidal function generated from a custom MATLAB script was used to fit this index distribution. The function required inputs of amplitude ( a ), shoulder location ( d 1 and d 2 in μm), and shoulder width ( w 1 and w 2 in μm), with the constraints maintained between 0 and 1: (2) y ( d ) = a 1 + e d − d 1 / w 1 + 1 − a 1 + e d − d 2 / w 2 . Lastly, quantifiable changes in the vasculature over 72 hours were recorded by measuring blood vessel diameter in ImageJ. The percent change in blood vessel (b.v.) diameter was calculated as the difference between vessel diameter at the current time point and vessel diameter at 2 hours divided by the vessel diameter at 2 hours post-insertion. Statistical Analysis A Welch’s t -test (unequal variance) was used to compare differences in processes extension, cell body migration, surface coverage, and morphological alterations between NG2 glia and microglia populations. To determine differences between time points within NG2 glia populations and blood vessel z-stacks, a repeated measures analysis of variance (ANOVA) was used. A p < 0.05 was chosen to demonstrate significant differences.

Supplementary Material supplement