2.1.

Animals and Pretreatments

Adult Sprague–Dawley male rats (250 to $300\mathrm{g}/\text{each}$, $n=62$) were divided into four different groups, as summarized in Table 1, in which some animals received daily intraperitoneal (i.p.) injections of 0.9% saline ($0.7\mathrm{cc}/100\mathrm{g}/\text{day}$) or cocaine HCl ($35\mathrm{mg}/\mathrm{kg}$) for consecutive 2 or 4 weeks, which were administered in their home cage. We chose a cocaine dose of $35\mathrm{mg}/\mathrm{kg}$ (i.p.) since this dose results in cocaine plasma levels consistent to those observed in cocaine abusers.¹³ In vivo imaging experiments were performed after 1-day withdrawal from pretreatments.

Table 1

Experimental design: animal groups, pretreatment and imaging approaches.

2.2.

Surgical Preparation for In Vivo Imaging

Rats were anesthetized and ventilated with 1.5% to 3% isoflurane mixed in pure oxygen during the surgery. A femoral artery was catheterized for continuous arterial blood pressure monitoring and a femoral vein from the same side was catheterized for drug administration. The rat was then positioned in a stereotaxic frame (KOPF 900) to minimize brain motion. A cranial window ($\sim 5\times 4{\mathrm{mm}}^2$) was created above the right somatosensory cortex (AP: $+2\mathrm{mm}$ to $-3\mathrm{mm}$; LR $+2\mathrm{mm}$ to $+6\mathrm{mm}$). After the dura was carefully removed, the exposed brain surface was immediately covered with 1.25% agarose gel and affixed with a $100\text{-}\mu \mathrm{m}$-thick glass coverslip using biocompatible cyanocrylic glue to maintain normal cranial pressure. During the surgery and the later imaging of the brain, the physiological state of the animal was continuously monitored, including electrocardiography, mean arterial blood pressure (MABP), respiration rate, and body temperature (Module 224002, Small Animal Instruments). All of the procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committees of Stony Brook University.

2.3.

In Vivo Optical Imaging

Figure 1 shows a multimodality optical imaging platform employed to acquire all the in vivo image data presented in this study, in which 3-D ODT and MW-LSI were integrated into a modified zoom fluorescence microscope (AZ100, Nikon) allowing for simultaneous imaging. For 3-D ODT, a fast spectral-domain OCT system illuminated with a broadband source ($\lambda =1.3\mu \mathrm{m}$, $\mathrm{\Delta }\lambda \ge 90\mathrm{nm}$; Inphenix) was integrated into the zoom microscope via a custom dichroic mirror (DM1) reflecting $\lambda >1\mu \mathrm{m}$. The collimated light beam ($\mathrm{\phi }5\mathrm{mm}$) exiting the sample arm of the OCT engine was transversely scanned by a pair of servo mirrors (VM500, Cambridge Tech.), focused by an achromate ($f40\mathrm{mm}/0.1\mathrm{NA}$), and reflected by DM1 onto the cortex. The backscattered light from brain was recombined with the reference light and detected by a high-speed linear spectrograph (a 1024-pixel InGaAs array; Goodrich). By synchronizing with servo mirrors, two-dimensional (2-D) OCT image ($z-x$ cross section) was acquired at up to 140 fps and 3-D OCT was acquired by additional $y$-axis scanning. The acquired OCT dataset were transferred to a solid-state drive on a workstation in which graphics processing unit accelerated computing enabled parallel image processing for real-time display of 2-D and 3-D images of both amplitude (i.e., OCT, OCA or optical coherence angiography) and phase (e.g., CBFv) distributions on the cortical brain. The axial resolution of $8\mu \mathrm{m}$ was determined by the coherence length $L_c=2{(\ln 2)}^{1/2}/\pi ·{\lambda }^2/\mathrm{\Delta }\lambda$ and the transverse resolution of $\sim \mathrm{\phi }12\mu \mathrm{m}$ was determined by the focusing optics ($f40\mathrm{mm}/0.1\mathrm{NA}$). A typical field of view (FOV) of $5\times 4\times 2{\mathrm{mm}}^3$ on the rat’s cortex was acquired; and specific raster scanning schemes (e.g., dense sampling along $x$-axis) were implemented to optimize flow detection sensitivity for 3-D ODT and simultaneous 3-D OCA imaging (vasculature). For MW-LSI, two light emitting diodes ($150\mathrm{mW}/\text{each}$) at the wavelengths of ${\lambda }_1=570\mathrm{nm}$ and ${\lambda }_2=630\mathrm{nm}$ were coupled into a $\mathrm{\phi }3\mathrm{mm}$ fiber bundle ($\mathrm{NA}/0.25$) for illumination of the exposed cortex to image the dynamical changes of total blood volume ($\mathrm{\Delta }[\mathrm{tHb}]$), deoxygenated hemoglobin ($\mathrm{\Delta }[\mathrm{HbR}]$), and thus oxygenated hemoglobin ($\mathrm{\Delta }[{\mathrm{HbO}}_2]=\mathrm{\Delta }[\mathrm{tHb}]-\mathrm{\Delta }[\mathrm{HbR}]$).^14,15 In addition, a pigtailed diode laser (60 mW) at ${\lambda }_3=830\mathrm{nm}$ was delivered through 1:3 monomode fiber couplers (NA/0.12) to a ring illuminator (C1) for LSI imaging. All three channels were pulse modulated via a time-base (time-sharing) for sequential “wavelength-multiplexed” imaging at up to 16 Hz. Synchronized with spectral illumination, the backreflection from the exposed cortex (e.g., a FOV of $5\times 6{\mathrm{mm}}^2$) in each channel was collected via the microscope optics ($2\times /0.22\mathrm{NA}$) and imaged by a 16-bit sCMOS camera (Zyla 5.5, Andor). Changes in [${\mathrm{HbO}}_2$] and [HbR] were calculated directly through the time-lapse images at ${\lambda }_1$ and ${\lambda }_2$;¹⁶ the LSI flow image series were reconstructed by computing the speckle variances in both spatial domain (i.e., calculating speckle variance within adjacent pixels in each frame) and temporal domain (i.e., across adjacent frames).¹⁷ The 2-D flow images by LSI were coregistered with 3-D CBFv images by ODT to map the absolute flow rates of individual vessels and identify their flow types (venous or arterial flows), but LSI was used to track fast dynamic features of the vascular trees after acute cocaine.

Fig. 1

A schematic diagram of a multimodality optical imaging platform that combines 3-D optical coherence tomography (3-D OCT) and multiple wavelength laser speckle contrast imaging (MW-LSI) used in this study. The left dashed box represents the MW-LSI, which consists of three alternatively switching light sources, e.g., a laser diode ${\lambda }_3=830\mathrm{nm}$ for CBFv imaging and two LEDs at ${\lambda }_{\mathrm{1,2}}=\mathrm{570,630}\mathrm{nm}$ for oxygenation imaging. The right dashed box is a 1310-nm OCT system. SM, single mode; CM, collimator; BBS, broadband source ($\lambda =1.3\mu \mathrm{m}$); LD, aiming laser ($\lambda =670\mathrm{nm}$); FPC, fiber-optic polarization controller. Left dash box: modified zoom microscope. C1, C2: epi-illumination cube 1, 2. DM1: dichroic beam splitter (${\lambda }_D=1\mu \mathrm{m}$); L1: $2\times \mathrm{APO}$ ($f=45\mathrm{mm}$, $\mathrm{NA}=0.22$).

During the in vivo studies, 3-D OCA and ODT images were acquired by the OCT system to evaluate the effects of chronic cocaine on vascular density and CBFv in the somatosensory cortex of rats in Group 1 (A–C) in Table 1. MW-LSI images were captured every 2 min starting 10 min before saline or cocaine injection (i.e., baseline) till 30 min postsaline or cocaine injection, so that the dynamic characteristics of cocaine or saline injection on CBF, [HbR] and [${\mathrm{HbO}}_2$] were derived for each rat in Group 4 (A–C).

2.4.

Ex Vivo Assessment of Microvascular Density in Cortex

To further study the effect of cocaine on vascular density, we used ex vivo fluorescence measurement to assess microvascular density in the brain of animals with or without chronic cocaine pretreatment. As listed in Group 2 (A–C) in Table 1, animals were sacrificed 24 h after pretreatment withdrawal of either saline ($0.7\mathrm{cc}/100\mathrm{g}/\text{day}$, i.p., Group 2A) or cocaine ($35\mathrm{mg}/\mathrm{kg}/\text{day}$, i.p.) given for 2 weeks (Group 2B) or 4 weeks (Group 2C). fluorescein isothiocyanate (FITC)-Dextran (mol wt $2\times {10}^6$, $50\mathrm{mg}/\mathrm{mL}$), a fluorescence dye, was intracardiacally infused ($500\mu \mathrm{L}$) at 1 min before the animal was euthanized. Then, the whole brain of the rat was removed from the skull, immediately immersed and incubated in cold 4% formaldehyde solution (Thermo Fisher Scientific, Waltham, Massachusetts) overnight at 4°C. The brain was then continually immersed and fixed with increasing sucrose ($\ge 99.5\%$, Sigma-Aldrich, St. Louis, Missouri) gradients from 10% to 20% and 30%. The prepared brain was then embedded in Tissue-Tek OCT (Thermo Fisher Scientific) and was cut into $\sim 10\text{-}\mu \mathrm{m}$ thick coronal slices at bregma $+3.20$ on the cryostat (Leica CM3050 S, Leica Biosystems, Richmond, Illinois). Five or more fluorescence images were taken for each brain slice in the region of interest (ROI) with a $20\times$ objective using a fluorescence microscope (E80i, Nikon Instruments).

2.5.

Assessment of Vascular Endothelial Growth Factor Histochemistry in Cortex

Animals from Group 3 were used for VEGF immunofluorescence assessment, including control animals (Group $3{\mathrm{A}}_0$ and ${\mathrm{A}}_1$) and animals with 2-week and 4-week cocaine pretreatment (i.e., Group 3B and Group 3C, respectively). After 24-h withdrawal from the pretreatment, each animal was perfused with 4% formaldehyde (Thermo Fisher Scientific) and brains were collected, postfixed in 4% formaldehyde solution overnight, and kept in 30% sucrose until they sank to the bottom of the tube. Then the brains were cryosectioned into $\sim 10\text{-}\mu \mathrm{m}$-thick coronal slices (Leica CM3050 S, Leica Biosystems). The brain slices were first blocked with 5% goat serum (ab 7481, Abcam) for 30 min. Then the samples were incubated with anti-VEGF antibody (ab 52917, Abcam) at 1:200 dilution for 1 h, followed by a further 1-h incubation with an Alexa Fluor^® 488-conjugated Goat Antirabbit IgG H&L (1:1000 ab150077, Abcam). The slices were finally mounted with 2-[4-(aminoiminomethyl)phenyl]-1H-indole-6-carboximidamide, dihydrochloride Fluoromount-G^® (SouthernBiotech, Birmingham, Alabama). All procedures were done at room temperature. The negative controls underwent all the procedures in parallel with other slices except that they were incubated in normal antibody dilution buffer ($\mathrm{PBS}+1\%$ normal goat serum $+0.2\%$ Triton X-100) instead of in the anti-VEGF antibody. All VEGF fluorescence images were acquired at the same exposure time with a Nikon E80i fluorescence microscope.

2.6.

Image Processing and Data Analysis

Reconstruction of 3-D OCA images for assessing the vascular density was computed based on a speckle variation approach using Hessian filtering,^18,19 and reconstruction of 3-D ODT image for assessing CBFv was based on Doppler flow reconstruction algorithms, such as phase subtraction method or phase intensity method to enhance minute flow detection.^8,20 To quantify size-dependent vasoconstriction induced by chronic cocaine, we divided the vessels in the FOV as small (${\mathrm{\phi }}_{\mathrm{S}}=0$ to $100\mu \mathrm{m}$), medium (${\mathrm{\phi }}_{\mathrm{M}}=100$ to $200\mu \mathrm{m}$), and large (${\mathrm{\phi }}_{\mathrm{L}}>200\mu \mathrm{m}$) vessels. The vascular density was quantified by the fill factor (FF), defined as the ratio of the number of pixels occupied by vessels to the total number of pixels within the selected ROI, i.e.,

For MW-LSI images, cocaine or saline induced changes, e.g., $\mathrm{\Delta }[{\mathrm{HbO}}_2]$ ($[{\mathrm{HbO}}_2]=[\mathrm{HbT}]-[\mathrm{HbR}]$) and $\mathrm{\Delta }[\mathrm{HbR}]$ were calculated directly through the time-lapse images at ${\lambda }_1$ (i.e., [HbT]) and ${\lambda }_2$ (i.e., [HbR]); the LSI flow image series were reconstructed by computing the speckle variances at ${\lambda }_3$ in both spatial domain (i.e., calculating speckle variance within adjacent pixels in each frame) and temporal domain (i.e., across adjacent frames).²¹ Several ROIs in avascular areas (avoiding of apparent vessels) were selected to compute tissue $\mathrm{\Delta }[{\mathrm{HbO}}_2]$ and $\mathrm{\Delta }[\mathrm{HbR}]$ at each time point.

To assess microvascular density from the ex vivo fluorescent images, we adopted the Otsu threshold selection method, an unsupervised segmentation algorithm which utilized discriminant analysis (e.g., measurement of separability) to evaluate the “goodness” of threshold and thus automatically select an optimal threshold.²² Briefly, Otsu’s method is based on the separation of graylevel class ${\mathrm{C}}_0$ (objects) from ${\mathrm{C}}_1$ (background) by a threshold $k$. To obtain the optimal threshold, the total variance of levels $\eta$:

For microvascular density analysis, the Otsu’s thresholding method was applied to each fluorescence image (grayscale image), converting it into a binarized image for further analysis by using ImageJ software for counting the vessel numbers within the FOV of the image. The variation of the threshold from image to image was small and negligible; thus, the same threshold was applied in the density quantification. The microvasculature density of a unit area was defined as the ratio of the vessel number over the actual size of the FOV ($0.6960\times 0.5200{\mathrm{mm}}^2$) in each area. To get the vessel density of each area from each animal, densities of five randomly chosen images were averaged.

VEGF levels were analyzed by calculating the percentage of VEGF fluorescent pixels over the total pixels within the ROI of the image to result in the % area of VEGF. Specifically, ImageJ was used to analyze all the VEGF fluorescently labeled images (grayscale images), with a preset threshold (T) defined by the averaged graylevel of multiple selected background ROI ($N=10$) to segment VEGF signal against its background noise:

The expression level of VEGF was calculated by taking the ratio of the pixels with the VEGF fluorescence over the total pixels of the ROI image (presented as % area). Three randomly chosen images of the somatosensory cortex from each rat were analyzed and averaged for statistical comparisons.

2.7.

Statistical Analysis

Data were presented as $\text{mean}\pm \mathrm{S}.\mathrm{D}$. and $p$-values were analyzed by performing paired $t$-test or one-way factorial analysis of variance (ANOVA) test (Systat software) to determine differences between groups. Significance was set at $p<0.05$ (double tail).

3.1.

Chronic Cocaine Decreased Cerebral Blood Flow Velocity Across the Cerebrovascular Tree

Figures 2(a) and 2(c) show CBFv images of the somatosensory cortex in control rats with saline pretreatment (0.9% saline, $0.7\mathrm{cc}/100\mathrm{g}/\text{day}$, i.p.) for 2 and 4 weeks, and Figs. 2(b) and 2(d) show the images for rats exposed to chronic cocaine ($35\mathrm{mg}/\mathrm{kg}/\text{day}$, i.p.) for 2 and 4 weeks. Compared with control animals, chronic cocaine exposure for either 2 or 4 weeks significantly decreased CBFv across the cerebrovascular network. Figures 2(e) and 2(f) summarize the quantitative comparisons of CBFv in large (${\mathrm{\phi }}_{\mathrm{l}}=293.40\pm 52.25\mu \mathrm{m}$), medium (${\mathrm{\phi }}_{\mathrm{m}}=124.27\pm 16.09\mu \mathrm{m}$), and small vessels (${\mathrm{\phi }}_{\mathrm{s}}=56.23\pm 13.85\mu \mathrm{m}$) from rats with or without cocaine pretreatment [Group 1(A–C), Table 1]. Specifically, after 2-week cocaine pretreatment (Group 1B, Table 1), basal CBFv decreased in large vessels from $15.24\pm 1.47\mathrm{mm}/\mathrm{s}$ in control rats to $11.58\pm 1.05\mathrm{mm}/\mathrm{s}$ ($p<0.001$); in medium vessels, it decreased from $14.26\pm 1.21\mathrm{mm}/\mathrm{s}$ to $10.80\pm 1.39\mathrm{mm}/\mathrm{s}$ ($p<0.001$); and in small vessels, it decreased from $12.45\pm 1.43\mathrm{mm}/\mathrm{s}$ to $8.34\pm 0.96\mathrm{mm}/\mathrm{s}$ ($p<0.001$). For the 4-week cocaine-exposed group (Group 1C, Table 1), basal CBFv in big vessels was reduced from $15.38\pm 1.35\mathrm{mm}/\mathrm{s}$ in control rats to $11.45\pm 1.34\mathrm{mm}/\mathrm{s}$ ($p<0.001$); in medium vessels, it was decreased from $13.81\pm 1.20\mathrm{mm}/\mathrm{s}$ to $10.63\pm 1.30\mathrm{mm}/\mathrm{s}$ ($p<0.001$), and in small vessels from $12.15\pm 1.50\mathrm{mm}/\mathrm{s}$ to $7.68\pm 0.91\mathrm{mm}/\mathrm{s}$ ($p<0.001$) [Fig. 2(f)].

Fig. 2

Chronic cocaine decreased CBFv across the cerebrovascular tree. Quantitative CBFv (ODT) images in the somatosensory cortex of the (a, c) saline-treated control groups versus (b, d) those of the chronic cocaine-treated groups for 2 and 4 weeks. Image sizes are $3.2\times 3.2{\mathrm{mm}}^2$, $\text{scale bar}=200\mu \mathrm{m}$. Yellow, green, and blue arrows are used to demonstrate the large ($\overline{\mathrm{\phi }}=293.40\pm 52.25\mu \mathrm{m}$; ${\mathrm{\phi }}_{\mathrm{L}}\ge 200\mu \mathrm{m}$), medium ($\overline{\mathrm{\phi }}=124.27\pm 16.09\mu \mathrm{m}$; $100\mu \mathrm{m}\le {\mathrm{\phi }}_{\mathrm{M}}<200\mu \mathrm{m}$), and small vessels ($\overline{\mathrm{\phi }}=56.23\pm 13.85\mu \mathrm{m}$; ${\mathrm{\phi }}_{\mathrm{S}}<100\mu \mathrm{m}$), respectively. (e, f) Statistical comparisons of CBFv among various groups, indicating the significant decreases of CBFv across the whole vascular trees. * significant differences between saline- and cocaine-treated groups ($n=5$, $m=12$; $*p<0.05$).

A comparison between the cocaine groups showed that CBFv in small vessels was significantly lower after 4-week than 2-week cocaine exposures, e.g., $7.68\pm 0.91\mathrm{mm}/\mathrm{s}$ versus $8.34\pm 0.96\mathrm{mm}/\mathrm{s}$, respectively ($p=0.016$). Therefore, these results show that chronic cocaine decreased CBFv across all blood vessel sizes in the cerebral cortex and that in small vessels, the decreases were larger after 4 weeks than 2 weeks of cocaine exposures.

3.2.

Chronic Cocaine Increased Density of Small Vessels in Cortex: In Vivo Measures

Figures 3(a) through 3(d) illustrate the skeletonized cerebral vasculature maps extracted from the OCA images [Figs. 3(e) through 3(h)] in the somatosensory cortex. Different colors were selected to represent vessels within three different diameter ranges, i.e., “blue” for large vessels (${\mathrm{\phi }}_{\mathrm{l}}>200\mu \mathrm{m}$), “red” for medium vessels (${\mathrm{\phi }}_{\mathrm{m}}=100$ to $200\mu \mathrm{m}$), and “white” for small vessels (${\mathrm{\phi }}_{\mathrm{s}}<100\mu \mathrm{m}$), and their vascular densities were quantified by the corresponding FFs, defined above in Eq. (1).

Fig. 3

Chronic cocaine increased the density of small vessels in the somatosensory cortex in vivo. Skeletonized vasculature maps of the rat somatosensory cortex among (a, c) saline-treated control groups and (b, d) chronic cocaine-treated groups for 2 and 4 weeks. Image sizes are $2.5\times 2.5{\mathrm{mm}}^2$ (scale bar: $200\mu \mathrm{m}$). “Blue,” “red,” and “white” illustrate the large (${\mathrm{\phi }}_{\mathrm{L}}\ge 200\mu \mathrm{m}$), medium ($100\mu \mathrm{m}\le {\mathrm{\phi }}_{\mathrm{M}}<200\mu \mathrm{m}$), and small (${\mathrm{\phi }}_{\mathrm{S}}<100\mu \mathrm{m}$) vessels, respectively. (e–h) Optical coherence angiography (OCA) of the rat somatosensory cortex. (i, j) Statistical comparisons of vascular density change elicited by chronic cocaine indicate that the vascular density of small vessels increased in both cocaine groups. In the 2-week cocaine group, the FF increased from $0.08\pm 0.01$ in control to $0.11\pm 0.01$ ($*p=0.008$). For the 4-week cocaine group, the density increased from $0.08\pm 0.003$ in control to $0.13\pm 0.02$ ($*p=0.009$). A significant difference was found between saline-treated and cocaine-treated groups, but no significant difference was found between 2-week and 4-week cocaine groups ($n=5$, $m=10$).

Figures 3(i) and 3(j) show the comparisons for FFs in large, medium, and small vessels between control rats ($n=6$ in total) with 2-week or 4-week saline pretreatment and 2-week [Fig. 3(i)] or 4-week [Fig. 3(j)] cocaine pretreatments. Vascular density of small vessels increased in both cocaine groups [Figs. 3(i) and 3(j)]. For the 2-week cocaine group, the FF increased $41.70\pm 10.41\%$ from $0.08\pm 0.01$ in control to $0.11\pm 0.01$ ($p=0.008$) and for the 4-week cocaine group, it increased from $0.08\pm 0.003$ in control to $0.13\pm 0.02$ ($p=0.009$). The differences between the 2-week (Group 1B) and the 4-week (Group 1C) cocaine groups were not significant ($p=0.242$). In contrast for the large and medium vessels, there were no significant differences in vascular density among control and 2-week [Fig. 3(i)] and 4-week [Fig. 3(j)] cocaine pretreatment groups.

3.3.

Chronic Cocaine Increased Microvascular Density and Vascular Endothelial Growth Factor Levels in the Cortex: Ex Vivo Measures

In parallel to in vivo image analyses of cocaine-induced vascular density changes, ex vivo histochemistry results are presented in Figs. 4(a)–4(d), which correspond to micrographs of the microvasculature labeled with FITC-Dextran fluorescence indicator in control rats ($n=8$, Group 2A, Table 1) and in rats pretreated with cocaine for 2 and 4 weeks ($n=6$, 2 weeks for Group 3B, $n=6$, 4 weeks for Group 2C, Table 1). Figure 4(e) compares the microvasculature densities in the somatosensory cortex between control and chronic cocaine rats. It indicates that microvasculature density, in which control rats were $269.26\pm 9.51/{\mathrm{mm}}^2$ for the 2-week saline and $287.84\pm 24.59/{\mathrm{mm}}^2$ for the 4-week saline, was significantly higher in rats exposed to cocaine corresponding to $372.92\pm 29.96/{\mathrm{mm}}^2$ for the 2-week exposures ($p<0.001$) and to $392.88\pm 23.21/{\mathrm{mm}}^2$ for the 4-week exposures ($p<0.001$). The difference between the 2- and 4-week cocaine groups did not reach significance ($p=0.226$).

Fig. 4

Chronic cocaine increased microvascular density and VEGF expression levels in the somatosensory cortex. Micrographs of the microvasculature (FITC-Dextran) in the somatosensory cortex (a, c) after saline pretreatment and (b, d) after chronic cocaine administration. (e) Statistical comparisons of microvasculature density ($n=6$, $m=30$, $*p<0.05$). Micrographs of VEGF expression in (f, h) saline-treated rats and (g, i) cocaine-treated rats. Scale bar: $50\mu \mathrm{m}$; (j) Statistical comparisons of VEGF expression in the somatosensory cortex among all groups ($n=4$, $m=12$; $*p<0.05$). *: significant differences between saline-treated and cocaine-treated groups.

Figures 4(f)–4(i) illustrate the VEGF fluorescence from the somatosensory cortex in control and chronic cocaine rats. It shows that VEGF expression was increased after chronic cocaine. For the 2-week cocaine group (i.e., Group 3B), VEGF fluorescence increased from $0.44\pm 0.05$ to $0.86\pm 0.21$ (% area of VEGF, $p=0.021$) and for the 4-week cocaine group (i.e., Group 3C, Table 1), it increased from $0.45\pm 0.13$ to $1.32\pm 0.30$ ($p=0.006$). Comparisons between the cocaine groups showed that VEGF levels were significant higher in the 4-week than the 2-week cocaine groups ($p=0.047$).

3.4.

Cortical Cerebral Blood Flow Volume was Transiently Decreased with Acute Cocaine Challenge and was Longer Lasting in 4-week Than 2-week Cocaine Exposed Rats

Figure 5 illustrates the dynamic CBFv maps in the somatosensory cortex obtained with MW-LSI from a different group of animals (Group 4, Table 1), in control (Group 4A) and 2-week (Group 4B) and 4-week cocaine rats (Group 4C). The grayscale images at the first of each row represent baseline CBFv maps ($t=0\min$), the color image series show the ratio images of CBFv at various time points (e.g., $t=2$, 8, 14, 20, and 26 min) after saline or cocaine challenge versus its baselines in order to track CBFv changes with time. For example, Figures 5(a0)–5(a5) show the CBFv changes in a control rat before (i.e., $t\le 0\min$) and after saline ($0.1\mathrm{cc}/100\mathrm{g}$, i.v.). The “green” tone across the FOV reflects the level of blood flow in tissue, which does not change after saline injection. Figure 5(a6) summarizes the time course of CBFv changes for control rats (Group 4A) in response to saline, showing no significant change in CBFv before and after saline administration.

Fig. 5

Acute cocaine abruptly decreased CBFv in somatosensory cortex of chronically cocaine-exposed rats. Dynamic CBFv maps in the somatosensory cortex of control rats with 2-week (red curve) and 4-week (blue curve) saline-treatment (a0–a5), and cocaine treated rats treated for 2 weeks (b0–b5) or 4 weeks (c0–c5) in response to saline ($0.1\mathrm{cc}/100\mathrm{g}$, i.v.) or acute cocaine ($1\mathrm{mg}/\mathrm{kg}$, i.v.). The first column presents the laser speckle contrast images taken at ${\lambda }_3=830\mathrm{nm}$, reflecting the CBFv changes in this area. (a6, b6, c6) Time courses of CBF changes in response to saline or cocaine among all groups, indicating an abrupt and transient CBFv decrease after acute cocaine in chronically exposed cocaine rats and this decrease was more dramatic and longer-lasting in the 4-week than the 2-week cocaine rats. *significant differences between saline- and cocaine-treated groups ($n=3$, $m=9$, $*p<0.05$).

Figures 5(b0) through 5(b5) illustrate the CBFv changes to an acute cocaine challenge ($1\mathrm{mg}/\mathrm{kg}$, i.v.) in a rat exposed to 2 weeks of cocaine (Group 4B, Table 1), where the “blue” tone reveals global transient CBFv decrease in somatosensory cortex till $\sim 14\min$ when it returns to baseline levels. Figure 5(b6) summarizes the time course of CBFv changes for the 2-week cocaine rats (Group 4B) exposed to acute cocaine, showing an abrupt decrease in CBFv within $t=2\min$ of cocaine injection (minimum decrease $-11.90\pm 2.60\%$; $p=0.02$) followed by a gradual recovery after 14-min postinjection.

Figures 5(c0) through 5(c6) illustrate the CBFv changes to an acute cocaine challenge ($1\mathrm{mg}/\mathrm{kg}$, i.v.) in a rat exposed to 4 weeks of cocaine (Group 4C, Table 1), showing also an abrupt CBFv decrease within $t=2$ to 6 min after cocaine injection (minimum $-18.76\pm 6.20\%$; $p=0.039$). The decline in CBFv was significantly longer lasting than for the 2-week cocaine pretreated animals and did not fully recover even after 30-min postcocaine injection [Fig. 5(c6)]. Specifically, in 2-week cocaine rats (Group 4B), CBFv recovered within $t=16-22\min$ to baseline levels (i.e., $\mathrm{CBFv}=1.00\pm 1.25\%$), whereas in 4-week cocaine rats (Group 4C), CBFv was still $-14.72\pm 2.07\%$ below baseline; the differences between the groups were significant ($p<0.001$).

3.5.

$\mathit{\Delta }{\mathit{HbO}}_{\mathsf{2}}$ and HbR in Somatosensory Cortex After Acute Cocaine Challenge in Chronic Cocaine Rats

Figure 6 shows the dynamic changes of [${\mathrm{HbO}}_2$] after saline ($0.1\mathrm{cc}/100\mathrm{g}$, i.v.) in control rats (Group 4A, Table 1) and after acute cocaine ($1\mathrm{mg}/\mathrm{kg}$, i.v.) in cocaine pretreated for 2-week (Group 4B, Table 1) or 4-week rats (Group 4C, Table 1). Saline injection did not change [${\mathrm{HbO}}_2$] [maps in Figs. 6(a0) through 6(a5) and time course in Fig. 6(a6)]. By contrast, acute cocaine in the 2-week exposed rats significantly decreased [${\mathrm{HbO}}_2$] ($-3.88\pm 0.57\%$; $p=0.008$) at $t=6\text{-}\min$ postcocaine [Figs. 6(b1) through 6(b3)] and [${\mathrm{HbO}}_2$] was $-0.35\pm 0.47\%$ within $t=16$ to 22 min, indicating that at that time, it had returned to baseline [Fig. 6(b6)]. In the 4-week cocaine group, decreases in [${\mathrm{HbO}}_2$] were significantly larger than for the 2-week group ($-4.45\pm 0.34\%$; $p=0.001$ within $t=6$ to 8 min) and longer-lasting (e.g., still $-2.93\pm 0.46\%$ within $t=16$ to 22 min postinjection) ($p<0.001$) and did not recover even after 30-min postinjection [Fig. 6(c6)].

Fig. 6

Acute cocaine decreased [${\mathrm{HbO}}_2$] in somatosensory cortex of chronically cocaine exposed rats. Dynamic changes of tissue [${\mathrm{HbO}}_2$] in the somatosensory cortex of control rats with 2-week (red curve) and 4-week (blue curve) saline-treatment (a0–a5), and the rats treated with chronic cocaine for 2 weeks (b0–b5) and 4 weeks (c0–c5) in response to saline ($0.1\mathrm{cc}/100\mathrm{g}$, i.v.) or cocaine ($1\mathrm{mg}/\mathrm{kg}$, i.v.). The first column presents the images taken at ${\lambda }_1=570\mathrm{nm}$, reflecting the concentration of total hemoglobin in tissue. (a6, b6, c6) Time courses of [${\mathrm{HbO}}_2$] changes in response to saline or cocaine among all groups. [${\mathrm{HbO}}_2$] did not fluctuate after a saline injection in control rats while a single dose of cocaine dramatically decreased [${\mathrm{HbO}}_2$] in cocaine exposed rats. The varying pattern of [${\mathrm{HbO}}_2$] was similar with that of CBFv. *significant differences between saline- and cocaine-treated groups ($n=3$, $m=9$, $*p<0.05$).

Figure 7 shows the comparison of the dynamic changes in deoxygenated hemoglobin concentration [HbR]. Again, saline injection did not change [HbR] in control rats [Figs. 7(a0) through 7(a6)], but acute cocaine triggered a significant [HbR] increase to $1.68\pm 0.14\%$ ($n=3$, $p=0.009$) at $t=6\min$ after cocaine injection in the 2-week and to $2.50\pm 0.54\%$ ($n=3$, $p=0.005$) at $t=8\min$ in the 4-week cocaine group and at $t=16–22\min$ [HbR] changes were $0.41\pm 0.19\%$ and $1.23\pm 0.28\%$. [HbR] returned to baseline after 20-min postcocaine in the 2-week group but in the 4-week group, [HbR] were still elevated over the baseline 28-min postcocaine.

Fig. 7

Acute cocaine increased [HbR] in somatosensory cortex of chronically cocaine exposed rats. Dynamic changes in tissue [HbR] of control rats with 2-week (red curve) and 4-week (blue curve) saline-treatment (a0–a5), and of rats treated with chronic cocaine for 2 weeks (b0–b5) and 4 weeks (c0–c5) in response to saline ($0.1\mathrm{cc}/100\mathrm{g}$, i.v.) or cocaine ($1\mathrm{mg}/\mathrm{kg}$, i.v.). The first column presents images taken at ${\lambda }_1=570\mathrm{nm}$, reflecting the concentration of total hemoglobin in tissue. (a6, b6, c6) Time courses of [HbR] variations in response to saline or cocaine among all groups. In contrast to the decrease of CBFv and [${\mathrm{HbO}}_2$], [HbR] increased in response to acute cocaine in cocaine pretreated rats. *significant differences between saline-treated and cocaine-treated groups ($n=3$, $m=9$, $*p<0.05$).