2.1.

Cell Cultivation

The C6 glioma cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), $2\mathrm{mM}$ L-glutamine, $50\mu \mathrm{g}∕\mathrm{mL}$ streptomycin, and $50\mathrm{U}∕\mathrm{mL}$ penicillin. Cells were maintained in an incubator with $5\%\mathrm{C}{\mathrm{O}}_2$ , 100% humidity at $37°\mathrm{C}$ . Before implantation to the rat brain, cells growing in an exponential rate were harvested by Trypsin for $5\min$ at $37°\mathrm{C}$ . Trypsin was inactivated by medium with 10% FCS, and the cells centrifuged at $600\mathrm{g}$ for $5\min$ . The pellets were then resuspended in RPMI 1640 medium without any supplement, at a concentration of ${10}^6\text{cells}∕\mu \mathrm{L}$ . Gentle manual agitation was applied to keep the cells in suspension before implantation.

2.2.

Implantation Surgery

Thirty adult male Sprague-Dawley rats ( $230–280\mathrm{g}$ , Southeast University vivarium) were used in this study. The animals were kept in their habitual environment untill the day of the experiment. All animals were anesthetized with 4% Nembutal ( $40\mathrm{mg}∕\mathrm{kg}$ , i.p.) and mounted into a stereotactic frame (incisor bar $2.4\mathrm{mm}$ below the interaural line) in a flat-skull position. After the periosteum was unmasked, two burr holes were performed with a $1\mathrm{mm}$ dental drill in the calvaria, respectively, on the left and right sides, $3\mathrm{mm}$ lateral from the midline, $1\mathrm{mm}$ posterior to the bregma. Suspension $\left(10\mu \mathrm{L}\right)$ with $1\times {10}^6$ C6 glioma cells were stereotactically injected at a depth of $5\mathrm{mm}$ , by a $25\mu \mathrm{L}$ microamount syringe (the syringe was pricked $6\mathrm{mm}$ depth, and then was drawn $1\mathrm{mm}$ back) on the right side. The same volumes of sodium chloride were injected at the same depth on the left side. The holes were sealed with bone wax, and the operative field washed with saline solution and the skin sutured. The body temperature was maintained at $37\pm 0.5°\mathrm{C}$ using a homeothermic heating pad throughout the experiment. All the experimental procedures follow the institutional guidelines and were approved by the Southeast University, China.

2.3.

NIRs and Experiments

NIR spectroscopy (NIRS) experiments were performed on POD 3, 10, and 17 with 10 rats each time. The homebuilt NIRs experimental system¹¹ includes a light source (HL-2000, Ocean Optics, Inc., Dunedin, FL), a bifurcated needle probe ( $600\mu \mathrm{m}$ o.d. with two single mode fibers of $100\mu \mathrm{m}$ ), a spectrometer (USB 2000, Ocean Optics, Inc.), step motor and its driver system, a laptop, as shown in Fig. 1 . The bifurcated needle probe is composed of two branches. One branch is connected to the light source and the other to the spectrometer. The backscattered intensity of light depends strongly on the absorption and scattering properties of the tissue. The optic fiber probe was held perpendicularly above the exposed calvaria surface and mounted on a step motor, which was attached to a stereotaxic frame. This step motor drives the fiber probe deep into the brain at an interval of $0.2\mathrm{mm}$ .

Fig. 1

Diagram of the NIRS system for measurements on brain tissue. $1–5\mathrm{W}$ halogen lamp as the light source. 2—Optic probe containing two silica fibers. 3—USB2000 spectrometer. 4—Computer and software. 5—MID-7604 driver. 6—PCI-7344 controller and step motor, a—the point of implant hole and the projection C6 glioma and b—symmetric point in the contralateral skull.

For data acquisition, LabView (National Instruments, Austin, TX) was used to program the interface between the spectrometer and the computer. The integration time, which was taken by the detector to read out the intensity of backscattered light, was kept constant at $100\mathrm{ms}$ during the entire set of measurements. The measurements were taken on the left and right brain. On each side, 40 steps in each brain side were measured, starting from the cortex with a spatial interval of $0.2\mathrm{mm}$ . The data were recorded for a period of $10\mathrm{s}$ at $2\mathrm{Hz}$ at each step.

2.4.

Calculating the Reduced Scattering Coefficient

The reduced scattering coefficient $\left({\mu }_{\mathrm{s}}^{\prime }\right)$ serves as an index for the light-scattering property of the tissue. In principle, the intensity of backscattered light highly depends on light-scattering features of the tissue. Johns ⁵ had found the slopes of reflectance spectra curves between 700 and $850\mathrm{nm}$ could be used to differentiate cerebral gray matter and white matter. Qian ¹¹ established a relationship among the reduced scattering coefficient, the profiles of the collected spectra from $700\text{to}850\mathrm{nm}$ , and the wavelength. In this study, an empirical formula between the reduced scattering coefficient and the slope between 700 and $850\mathrm{nm}$ was deduced by simulation experiment for the NIRs system before the rat experiments.

An intralipid solution of 8, 6, 4, 2, and 1% concentrations were chosen in the simulation experiment because these solutions yield light-scattering properties similar to those found in human tissues. The reduced scattering coefficients of these tissue simulation solutions (intralipid) were acquired by a standard Oximeter (model no. 96208, ISS, Inc., Illinois) working at 690 and $834\mathrm{nm}$ . At the same time, the reflectance spectra of these solutions were collected by the fiber-optic spectrometer of the NIRs system. Figure 2 shows the slopes and the relative reduced scattering coefficients.

Fig. 2

The reduced scattering coefficients from tissue simulation solution (intralipid) at 690 and $834\mathrm{nm}$ are shown with their slopes calculated from collected spectra of the same solutions. The coordinates of the $y$ axis are ${\mu }_{\mathrm{s}}^{\prime }$ , and the coordinates of the $x$ axis are the slopes.

The relationship between the slopes and the reduced scattering coefficients is obtained by the data fitting. Thus, an empirical formula for calculating the reduced scattering coefficient is derived as

Table 1

The reduced scattering coefficients of intralipid solution measured by Oximeter and calculated by Eqs. 1, 2 and their differences.

The accuracy of Eqs. 1, 2 were further validated through the solid-tissue phantom experiment. Fifteen grams of Gelatin powder (Sigma, St Louis, MO, USA) made from porcine skin were added into $200\mathrm{mL}$ of boiling water and was dissolved completely by stirring. After the solution was cooled down, the intralipid solution with a certain concentration was added to the prepared gelatin solution. After that, the solution was frozen for the formation of the gelatin phantom. The percentage concentration of the intralipid used was varied, depending on the required ${\mu }_{\mathrm{s}}^{\prime }$ . Five solutions with intralipid concentrations of 1–4% were used to create different ${\mu }_{\mathrm{s}}^{\prime }$ values for gelatin phantoms, whose ${\mu }_{\mathrm{s}690}^{\prime }$ and ${\mu }_{\mathrm{s}834}^{\prime }$ measured by the Oximeter and calculated by Eqs. 1, 2 and their differences are shown in Table 2 .

Table 2

The reduced scattering coefficients of gelatin phantoms measured by Oximeter and calculated by Eqs. 1, 2 and their differences.

It shows in Tables 1, 2 that the maximum difference is $3.1{\mathrm{cm}}^{-1}$ and mean difference at $690\mathrm{nm}$ is bigger than at $834\mathrm{nm}$ . The maximum difference at $834\mathrm{nm}$ is $2.5{\mathrm{cm}}^{-1}$ . By combining the specific fiber-optic spectrometer with the empirical equations, an efficient method for real-time determination of the tissue reduced scattering coefficient is established.

2.5.

Magnetic Resonance Imaging (MRI) and Histology Studies

MRI exams were performed at POD 10 and 17 on a 1.5-T MRI scanner (Philips Eclipse) using a human wrist coil after NIRs measurement. Gd-DTPA was applied intravenously at concentrations of $0.2\mathrm{mmol}∕\mathrm{kg}$ , $5\min$ before the scan. During the MRI scan, the rats were positioned in a plastic holder and anesthetized by 4% Nembutal ( $40\mathrm{mg}∕\mathrm{kg}$ , i.p.). Standard multislice sagittal and cross images were obtained by a TSE sequence with $\mathrm{TR}=4113\mathrm{ms}$ , $\mathrm{TE}=96\mathrm{ms}$ , $\mathrm{NSA}=2$ , $\mathrm{FOV}=13\mathrm{cm}$ , slice $\text{thickness}=3.0\mathrm{mm}$ , slice $\text{gap}=0.5\mathrm{mm}$ , $\text{resolution}=256\times 384$ , and seven slices.

After MRI, the rats were sacrificed with an anesthetic overdose and their brains were removed after the experiments. The brains were fixed in 10% formalin for more than $24\mathrm{h}$ and embedded in paraffin; consecutive $5\mu \mathrm{m}$ coronal sections were cut and stained with hematoxylin and eosin. A semiquantitative assessment of pathology was carried out by the neuropathologist for each individual specimen.

2.6.

Statistical Analysis

The data reported here consist of only the reduced scattering coefficient at 690 and $834\mathrm{nm}$ . Spectral information between 700 and $850\mathrm{nm}$ for each sampling at each location in the brain was acquired. Data were collected 20 times at each measured point in a $10\text{-}\mathrm{s}$ acquisition period and then averaged. The ${\mu }_{\mathrm{s}}^{\prime }$ data finally used in this study were the data at each point. For each side of a rat brain, there were 40 values because the probe went $8\mathrm{mm}$ deep into the brain at an interval of $0.2\mathrm{mm}$ . The ${\mu }_{\mathrm{s}}^{\prime }$ values were also averaged on 10 rats at each point of each side for POD 3, 10, and 17. Furthermore, all the 30 C6 glioma models were confirmed by the MRI and histology.

3.1.

Spectra and Light Scattering

Representative traces for the right brain (glioma) measured by the NIRS are shown in Fig. 3 . They represent the different spectral value at different depths. The reduced scattering coefficients $\left({\mu }_{\mathrm{s}}^{\prime }\right)$ at 690 and $834\mathrm{nm}$ were calculated by Eqs. (2.1) and (2.2) using these spectral slopes.

Fig. 3

Reflectance spectra in vivo obtained from right side on POD 10 during experiment from 1 to $8\mathrm{mm}$ deep within the brain. Open square represents $1\mathrm{mm}$ deep. Filled square represents $2\mathrm{mm}$ deep. Open triangle represents $3\mathrm{mm}$ deep. Filled triangle represents $4\mathrm{mm}$ deep. Open diamond represents $5\mathrm{mm}$ deep. Filled diamond represents $6\mathrm{mm}$ deep. Open circle represents $7\mathrm{mm}$ deep. Filled circle represents $8\mathrm{mm}$ deep.

Analysis of ${\mu }_{\mathrm{s}}^{\prime }$ showed that there were significant differences between left (control) and right (glioma) sides of the brain tissue (Fig. 4 ) on POD 10 and 17. Data in Fig. 4 are the average data at each point $(n=20)$ . ${\mu }_{\mathrm{s}690}^{\prime }$ versus depth have the same trend as ${\mu }_{\mathrm{s}834}^{\prime }$ versus depth. It shows that ${\mu }_{\mathrm{s}}^{\prime }$ of the right side within some range is lower than that of the left side at its corresponding position, and ${\mu }_{\mathrm{s}}^{\prime }$ of POD 17 is lower than of POD 10 in the right side. On POD 10, there was significant difference between the left and right from a depth of $3.2\text{to}6.8\mathrm{mm}$ . On POD 17, the difference between the left and right is from a depth $1.2\text{to}7.2\mathrm{mm}$ . The ${\mu }_{\mathrm{s}}^{\prime }$ values measured for the left (control) and right (glioma) brain side on POD 10 and 17 at depths of $1–8\mathrm{mm}$ are listed in Table 3 as $\text{mean}\pm \mathrm{SEM}$ , respectively.

Fig. 4

${\mu }_{\mathrm{s}}^{\prime }$ values for the left (control) and right (glioma) side from $0.2\text{to}8\mathrm{mm}$ deep within the brain. Open diamond represents $690\mathrm{nm}$ of the left side. Filled diamond represents $834\mathrm{nm}$ of the left side. Open square represents $690\mathrm{nm}$ of the right side on POD 10. Filled square represents $834\mathrm{nm}$ of the right side on POD 10. Open triangle represents $690\mathrm{nm}$ of the right side on POD 17. Filled triangle represents $834\mathrm{nm}$ of the right side on POD 17.

Table 3

The μs′ values for the left and right side on POD 10 and 17 at different deep within the brain (n=10) .

3.2.

MRI Imaging

In images of T2-weighted spin-echo and gradient-echo with Gd-DTPA enhancement, there were ellipse high signals in the right hemisphere, and these high signals were inhomogeneous in some rats (Fig. 5 ).

Fig. 5

(a) Emerge low signals in the center of high signals, which represent necrosis or hemorrhage. (b) shows inhomogeneous high signal in the position where implant C6 glioma cells on POD 17.

3.3.

Histology

The histopathological alterations of C6 gliomas were shown in Fig. 5. On a semiquantitative grading scale, all specimens had been classed into II/III grade by a neuropathologist. Prominent mitotic activity was observed under microscope on POD 10 [Fig. 6a and 6b ], by which gliomas are classed into grade II. An amount of tumor cells necrosis and hemorrhage in the central region of tumors occurred on POD 17 [Fig. 6c and 6d], by which gliomas are classed into grade III.

Fig. 6

Microphotographs of C6 gliomas. A, C, $\mathrm{D}\times 200$ E-H; $\mathrm{B}\times 400$ E-H. (a) Shows grade I glioma (POD 10); (b) a mitotic phenomenon in the center of the photograph (arrows); (c) grade III, shows necrosis at low right; (d) grade III, hemorrhage in right side (POD 17).